4.22 Viral DNA Packaging Motors

4.22 Viral DNA Packaging Motors CL Hetherington, University of California, Berkeley, CA, USA JR Moffitt, University of California, Berkeley, CA, USA and Harvard University, Cambridge, MA, USA PJ Jardine, University of Minnesota, Minneapolis, MN, USA C Bustamante, University of California, Berkeley, CA, USA; Lawrence Berkeley National Laboratory, Berkeley, CA, USA, and Howard Hughes Medical Institute, Chevy Chase, MD, USA r 2012 Elsevier B.V. All rights reserved.

4.22.1 4.22.2 4.22.2.1 4.22.2.2 4.22.2.3 4.22.2.4 4.22.2.5 4.22.3 4.22.3.1 4.22.3.2 4.22.4 4.22.4.1 4.22.4.1.1 4.22.4.1.2 4.22.4.1.3 4.22.4.2 4.22.4.2.1 4.22.4.2.2 4.22.4.2.3 4.22.4.3 4.22.4.3.1 4.22.4.3.2 4.22.4.3.3 4.22.4.3.4 4.22.4.4 4.22.4.4.1 4.22.4.4.2 4.22.4.4.3 4.22.4.5 4.22.4.5.1 4.22.4.5.2 4.22.4.5.3 4.22.4.5.4 4.22.4.5.5 4.22.5 4.22.5.1 4.22.5.2 4.22.5.3 4.22.5.4 4.22.6 4.22.6.1 4.22.6.2 4.22.6.3 4.22.7 4.22.7.1 4.22.7.2 4.22.7.3 References

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Packaging in the Double-Stranded DNA Bacteriophages What are the Components and Organization of the Packaging Complex? Genome Recognition Element ATP Hydrolyzing Protein Oligomer RNA Oligomer Portal or Connector Capsid How is Packaging Initiated? Initiation via Terminal Protein Initiation via Terminase Small Subunit How is the DNA Translocated? Single-Molecule Measurements of Velocity of Packaging Packaging velocity depends on force – Thermodynamics of packaging Velocity depends on substrate – Identifying mechanical and chemical transitions The role of sequence motifs in force production High-Resolution Observations of Packaging Base-pair-resolution measurements reveal step size and coordination of stepping High-temporal-resolution measurements reveal coordination of ATP binding Thermodynamic efficiency Observations of Packaging on Modified Substrates Backbone phosphates provide processivity and regulate the chemical cycle Non-nucleic-acid-specific contacts drive translocation Motor–DNA interaction changes during the cycle The origin of the 2.5-bp step Defining the Mechanistic Role of the Different Components The ATPase may engage and drive the DNA The connector/portal is intimately involved in translocation and regulates the ATPase Putative connector/portal rotation Structural Models of DNA Translocation ATP binding Conformational changes coupled to ATP hydrolysis DNA binding Intersubunit coordination Constructing a molecular model for the packaging motor of f29 What are the Organization and Physics of the Genome during and after Packaging? Dynamics of Packaging at High Internal Filling Capsid Dynamics at High Internal Filling Energetics of DNA Confinement Organization of Packaged DNA How is Packaging Terminated? Termination of Packaging of Unit-Genome DNA Sequence-specific Termination of Packaging of Concatemeric DNA Headful Termination of Packaging of Concatemeric DNA Conclusions and Future Directions How Do Structural Features Dictate and Shape Structural Dynamics? How General are the Structural Dynamics in the Packaging Motors? Concluding Thoughts

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Viral DNA Packaging Motors

Abbreviations ASCE Additional Strand Conserved E NDPs nucleoside diphosphates

Glossary Additional Strand Conserved E fold A protein fold that is broadly conserved among many NTPase proteins in the cell. This fold is characterized by secondary structure – a sixstranded beta sheet – as well as by certain amino acid motifs such as Walker A and B, arginine finger, and catalytic glutamate. Many proteins containing this fold are molecular motors. Arginine finger This highly conserved motif of ASCE proteins is involved in coupling changes in nucleotide state to global conformational changes. The arginine finger of one subunit of an oligomeric ATPase will in many cases participate in the ATP-binding pocket of another subunit, hence providing a conduit for communication between subunits. ATPase An enzyme that catalyzes the hydrolysis of ATP into ADP and Pi, often coupled to another mechanical or biochemical function. A subset of the NTPases, which catalyze the hydrolysis of nucleotide triphosphates. Bacteriophage A virus that infects a prokaryote. Capsid The shell of a virus, composed of copies of one or more structural subunits arranged as a closed crystalline surface. Connector or Portal The protein channel through which DNA passes into the capsid during packaging and through which DNA exits during infection. The connector/portal is a dodecameric ring that replaces one fivefold vertex of the capsid. Cryo-electron microscopy 3-D reconstruction, or cryoelectron tomography A technique for nanometerresolution 3-D imaging of materials based on repeated electron microscope projections at different angles. The sample is flash-frozen at liquid nitrogen temperatures, allowing the study of objects in a nearly native state. Distance to mechanical transition state A quantity that helps describe the energy landscape for a mechanical reaction. It is the physical distance, along the mechanical reaction coordinate, between the ground state and the transition state for a mechanical step. DNA translocase A molecular motor that transports DNA.

4.22.1

Packaging in the Double-Stranded DNA Bacteriophages

As we have seen elsewhere in this volume, the cell in some ways resembles a factory, containing a coordinated network of specialized reaction centers, each executing specific tasks, connected by well-defined transport pathways. In this emerging picture of the cell, mechanical processes are ubiquitous and essential. But the machines that perform mechanical work are not limited to cells. Indeed, a critical task during the selfassembly of some viruses – nucleic acid packaging – is performed by a mechanical motor with close homology to

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NTPs nucleoside triphosphates TIRF total internal reflection fluorescence

Michaelis–Menten kinetics A model that explains the macroscopic kinetics of an enzyme-catalyzed reaction. The relationship between substrate concentration [S] and turnover rate v is given by v ¼ vmax Km½S þ½S where vmax is the maximum turnover rate and Km is the substrate concentration at which v ¼ 1/2 vmax. The simplest model k1

that fits this expression is E þ S " ES-kcat E þ P, but a large k1

class of more complicated models with show the same general form where Km and vmax are functions of the microscopic rate constants {ki}. Packaging motor The complex of molecules responsible for translocating DNA into the viral capsid during the assembly process of dsDNA bacteriophages. Ring ATPase An ATPase that functions as an oligomeric ring, often but not exclusively encircling the substrate on which it performs mechanical work. A common topology among molecular motors. Stall force A quantity that partially describes the energy landscape of a molecular motor. It is the load force at which the motor’s velocity drops to zero. The stall could result from thermodynamic considerations or from the advent of force-stimulated off-pathway processes. Terminase A protein hetero-oligomer responsible for packaging initiation and translocation in some phages. The terminase small subunit binds to specific initiation and cleavage sequences in the viral genome. The large subunit is an ATPase that translocates DNA and cleaves it in a manner regulated by the small subunit. Walker A and B motifs Two motifs highly conserved throughout the ASCE family of proteins, thought to position the nucleotide phosphate groups and a magnesium ion in preparation for hydrolysis. X-ray crystallography A method for A¨ngstrom-resolution 3-D determination of molecular structure based on modeling the diffraction pattern of a crystal composed of the molecule of interest. This powerful technique provides the highest-resolution information on fixed states of a biomolecule.

cellular proteins. In this chapter, we will focus on the function and mechanism of the packaging motor of double-stranded DNA bacteriophages. The insights gained from the examination of the packaging motor will shed light on the processes carried out by homologous proteins in the cell. Viruses, the most abundant biological entities on the planet,1 protect their genetic material inside of a protein shell. Formation of the viral particle occurs via one of two pathways. In some viruses, assembly of the viral shell (capsid) is nucleated by specific sites on the genome, and capsid assembly and genome condensation occur essentially together. In contrast, other viruses, including the majority of prokaryotic viruses,2

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first assemble a purely proteinaceous capsid, devoid of nucleic acid, and then physically pump their genetic material into this empty shell. This energetically unfavorable task is accomplished by a remarkable molecular machine called the packaging complex, which is comprised of several components that coordinate the conversion of chemical energy from nucleoside triphosphates (NTPs) into the needed mechanical work. Among the strongest and most efficient molecular motors known, the packaging motors have been subjected to four decades of research, and in the past years a variety of cutting-edge structural, biochemical, and single-molecule ‘in singulo’ methods have greatly refined our understanding of principles underlying the mechanism of these complex motors (reviewed in Refs. 3–9). The tailed bacteriophages utilize broadly similar structural and mechanistic elements to translocate double-stranded DNA into the capsid (Figure 1). In the interior of an infected cell, the viral genome is both replicated and expressed, leading to the production of multiple copies of the viral genome and viral ‘proheads.’ The proheads consist of an empty shell, called the ‘capsid,’ and a unique dodecameric structure that encloses a channel into the capsid, called the ‘portal’ or ‘connector.’ At initiation of packaging, a viral genome is specifically recognized by the packaging machinery, via either a ‘terminal protein’ or a ‘terminase small subunit,’ and associates with a capsid. The packaging motor then translocates the DNA into the capsid while ATP is hydrolyzed by an ATPase oligomer. The packaging complex consists of several obligate coaxial rings

which encircle and possibly engage the DNA: a ring of ATPase proteins, called either the ‘packaging ATPase’ or the ‘terminase large subunit;’ the dodecameric portal/connector; and, in the f29-like phages, a pentameric ring of RNA molecules called the ‘pRNA’ that bridges the ATPase to the portal/connector. At the conclusion of packaging, the ATPase and pRNA rings dissociate from the prohead and are replaced by a ‘tail,’ which stabilizes the complete and mature particle and later serves to inject the genome into the next target cell. The in vitro bacteriophage assembly process is remarkably well synchronized and efficient, with nearly 100% of viral genome copies being packaged for some species.5 Such efficiency has allowed the structural and dynamic details of packaging to be probed with a number of advanced techniques, such as cryoelectron microscopy 3-D reconstruction, X-ray crystallography, and single-molecule manipulation and fluorescence. Moreover, the study of viral packaging has itself catalyzed the development of new experimental and theoretical tools which have or will find a wide variety of biological applications.10–18 These techniques have been employed to answer a number of fundamental questions about the nature of viral packaging. In this chapter, we will focus on packaging by the doublestranded DNA bacteriophages due to the extensive body of literature – especially single-molecule reports – but the reader can be assured that packaging by some RNA viruses, while quite distinct from dsDNA packaging, is no less fascinating.2,9 We will address the outstanding issues in the study of DNA packaging, with particular emphasis on elucidating the mechanism of DNA

29-like phages Capsid

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Figure 1 Outline of viral packaging pathways, components not drawn to scale. (a) Bacteriophage f29 DNA, labeled with the terminal proteins (orange balls), forms a lariat and is bound by the ATPase (blue) as well as a prohead, composed of the capsid (yellow), connector (green at base of capsid), and pRNA (magenta). Packaging proceeds from the left terminal protein until the right terminal protein is reached. The ATPase and pRNA then dissociate and the tail complex (gray) attaches. (b) Terminase phages produce a DNA molecule with several concatenated genomes containing multiple recognition sequences (orange boxes). The terminase complex, composed of the large subunit (blue) and small subunit (red), binds to the sequences, cleaves, and associates with the prohead. After one genome has been packaged, the large subunit cleaves the DNA, dissociates from the capsid, and binds to a new prohead. The tail complex (gray) assembles on the packaged prohead.

Viral DNA Packaging Motors

translocation. We will highlight the advances and insights provided by the past decade of dynamic and structural research, and focus especially on the unique discoveries of single-molecule experiments. The topics we will cover here are: 1. What are the essential components of the packaging machinery, and what is the structure of the active complex? 2. How is packaging initiated? 3. How is DNA translocated? Specifically, how is the energy released by cycles of ATP hydrolysis converted into conformational changes of the packaging motor? How are those conformational changes coupled to mechanical work on the DNA? How are the multiple motor subunits coordinated? What components directly engage and perform work on the DNA? 4. What are the organization and physics of the genome during and after packaging? 5. How is packaging terminated? A variety of answers have been offered to these questions through both experimental and theoretical methods. In many cases, ambiguities still exist and future research will definitely be needed; however, for some of these questions, clear pictures are starting to emerge.

4.22.2

What are the Components and Organization of the Packaging Complex?

Different tailed bacteriophage species, with little or no sequence identity and targeting highly different hosts, employ functionally and structurally similar macromolecular tools to translocate dsDNA (reviewed in Refs. 4, 19). The universal elements of this toolkit – the ATPase and the connector/portal – appear to be modular, having been assorted independently according to phylogenetic studies.20 Hence, it is likely that determination of the structure or mechanism of one component in one viral species will be highly relevant for its role in other species. In this section, we will summarize the essential components of the packaging complex and review what is known about its organization (see Figure 1).

4.22.2.1

Genome Recognition Element

Efficient in vivo viral assembly requires that the replicated viral genomes be selected out of a background of cellular DNA. In

Connector

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the dsDNA bacteriophages, this is accomplished in two distinct ways. f29-like phage DNA is labeled at each end with a terminal protein during protein-primed replication, and the terminal proteins are specifically recognized by the packaging complex (reviewed in Refs. 21, 22). Because packaging proceeds from one end of the DNA to the other (conventionally termed left-to-right packaging), the left end terminal protein enters the capsid at the beginning of packaging and does not appear to interact with the packaging motor after initiation; the protein at the distal right end likely does not interact with the packaging motor until termination. In contrast, the terminase phages employ a protein called the terminase small subunit to recognize and bind a short viral DNA sequence and recruit the remainder of the complex (reviewed in Ref. 7). Sequence recognition is performed by a N-terminal winged helix-turn-helix domain23,24 as part of an eight- or ninemembered ring;24–27 the small subunit oligomer is thought to bind coaxially to a ring of large subunit molecules25 which can cleave the small subunit-associated DNA.28 During packaging, the DNA passes through the pore of the putative small subunit ring, then through the prohead-bound large subunit ring.24,29–31 The terminase small subunits remain bound to the packaging complex throughout packaging and, in some phages, direct cutting of the viral genome from a concatemeric DNA molecule during termination.

4.22.2.2

ATP Hydrolyzing Protein Oligomer

The packaging ATPase forms an oligomer that associates with the genome recognition element, with the DNA, and with the prohead (Figure 2) (reviewed in Ref. 7). The DNA is then translocated into the capsid while NTPs, typically but not exclusively adenosine triphosphate (ATP), are hydrolyzed into nucleoside diphosphates (NDPs) and inorganic phosphate (Pi). Sequence analysis has shown that the tailed bacteriophage packaging ATPases belong to the Additional Strand Conserved E (ASCE) division of ATP-hydrolyzing proteins, a large protein group responsible for a wide variety of cellular activities20,32,33 (Figure 3). The ASCE core is characterized by its secondary structure – a sheet formed of alternating beta strands and alpha helices – and by the amino acids found in certain highly conserved motifs such as Walker A, Walker B, catalytic glutamate, sensor, and arginine finger, all of which are required for the ATP binding and hydrolysis cycle. The details of these and other motifs in the packaging motors will be Capsid

pRNA

ATPase Figure 2 Structure of the f29 packaging complex. The dodecameric connector (cyan), with a channel large enough to permit dsDNA, is ensconced in the capsid (gray). The pentameric pRNA (magenta) binds the connector at one end and the ATPase (blue) at the other. Reprinted from Morais, M. C.; Koti, J. S.; Bowman, V. D.; Reyes-Aldrete, E.; Anderson, D. L.; Rossmann, M. G. Defining molecular and domain boundaries in the bacteriophage f29 DNA packaging motor. Structure 2008, 16(8), 1267–1274. Figure courtesy of M. Morais and M. Rossmann.

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R Figure 3 Phylogenetics of the ASCE division. Topology showing the highest-order relationships proposed by Burroughs, Iyer, and Aravind.20,32,33 Strands and helices comprising the ASCE core are shown and numbered; important ATP binding motifs are shown in magenta (A ¼ Walker A, B ¼ Walker B, R ¼ arginine finger). If a motif is not shown for one family, it has not been conclusively located. Where two identical motifs appear in one family, the motif has been found in both places. The RecA family includes the ssRNA packaging phage ATPases. The other families noted in this figure are: PilT, the pilus retraction ATPase; RecA, the filament-forming oligomeric ATPase involved in homologous recombination; SF1/SF2 helicases, non-ring-forming ATPases that manipulate nucleic acids; ABC, the ATP-binding cassette transmembrane transporters; KAP, a largely uncharacterized phyletic group likely to function in assembly of membrane-associated signaling complexes; STAND, another poorly understood group that participates in signal transduction during programmed cell death; AAA þ , ATPases associated with various cellular activities. Figure inspired by Burroughs, A. M.; Iyer, L. M.; Aravind, L. Comparative genomics and evolutionary trajectories of viral ATP dependent DNApackaging systems. Genome Dyn. 2007, 48–65, with permission from L. Aravind. Copyright by S. Karger AG, Basel.

discussed below in Section 4.22.4.1.3 on the role of motifs in force generation. In terminase viruses, the terminase large subunit is the packaging ATPase; it is composed of two domains – an N-terminal ATPase domain, and a C-terminal nuclease domain.20,34 X-ray crystallography of the T4

terminase large subunit has confirmed the presence of a RecAlike canonical ATP-binding fold but indicates some degree of divergence from the other RecA-like proteins.5,28 In contrast, the f29-like bacteriophages – as well as some eukaryotic viruses such as the poxviruses – utilize an ATPase closely

Viral DNA Packaging Motors related to the HerA/FtsK family of dsDNA translocases.20 The sequence homology of the viral ATPases to known molecular motors strongly suggests that these ATPases are also responsible for DNA translocation. Packaging ATPases, like many ASCE proteins, take the form of an oligomeric ring in the packaging complex (Figure 2). The exact oligomeric state may vary between phage types: five subunits are visible in cryo-electron microscopy 3-D reconstruction (cryo-EM) of f2936 and T4,28 whereas l may have four subunits.25 The pentameric packaging motor of f29 has a different oligomeric state than its hexameric cousins in the HerA/FtsK family, further suggesting that the exact number is not crucial for DNA translocase activity. This circular geometry may have important functional consequences such as enabling communication and coordination between the subunits, as will be discussed in the translocation section of this chapter (Section 4.22.4). The terminase ATPase domain is covalently linked to a C-terminal nuclease containing a RNAse H fold.20,28,34,37,38 This nuclease domain also contains an ATPase motif which may be used exclusively for slow-turnover, low-affinity cleavage,37,39,40 although there is some indication that nuclease activity depends on the Walker B site in the ASCE domain.39,41 In addition to cleavage of the concatamer upon completion of packaging, the nuclease may play a role in supporting packaging itself. DNA-stimulated ATPase activity by the ATPase domain is strongly dependent on the presence of the nuclease domain.42 Also, in structural studies, the nuclease domain of both T4 and l appears to make direct contact with or bind the prohead (reviewed in Refs. 6, 43) so it is likely that the nuclease domain is structurally important for assembly or stabilization of the packaging complex. Finally, the crystal structure of T4 suggests several possible locations in the nuclease domain which could bind DNA during translocation rather than during cleavage.28

4.22.2.3

RNA Oligomer

The f29-like bacteriophages contain a unique factor not yet found in other packaging complexes: the prohead RNA, or pRNA (reviewed in Refs. 5, 44). pRNA originates as a 174-base transcript from the left end of the viral genome. After transcription, the pRNA oligomerizes on the prohead45 in the final step of morphogenesis, with no requirement for the presence of the ATPase. According to biochemical and cryo-EM studies, the pRNA oligomer is positioned as a bridge between the ATPase ring and the capsid (Figure 2).36,46–49 Functionally, pRNA helps to initiate packaging on the left end of the DNA,50,51 stimulates hydrolysis by the ATPase,50 and is required for assembly and activity of the packaging motor. After the completion of packaging, the pRNA, along with the associated ATPase, is released as the tail components are added to complete the mature viral particle.52 The positioning of the pRNA relative to the capsid and the ATPase suggests that it plays a structural role in the qcomplex. This hypothesis is borne out by cryo-EM reconstructions which show that the major helix of the pRNA extends down from the connector, interdigitating with and grasping the subunits of the ATPase pentamer.36

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Furthermore, sequence mutations to the helix that do not change its geometry have little effect on packaging, unless they simultaneously abolish ATPase binding. Finally, mutations which change the angle of the helices can modulate packaging efficiency.53,54 However, the pRNA may not be a static scaffold; some experiments suggest that flexibility at specific points in the pRNA is crucial for packaging.5,44 This flexibility may be required by some models of DNA translocation, to be described below, which involve large (nanometer-scale) conformational changes of the ATPase ring28 and possibly the connector49 as they engage and move the DNA. The pRNA, providing structural support for the ATPase, may itself need to adjust and reorient to accommodate or even induce such conformational changes. As it is likely that the pRNA plays an important role in organizing and positioning the ATPase, much effort has been devoted to determining the organization of the pRNA oligomer (reviewed in Refs. 5, 44). A set of clever experiments utilized the fact that compensatory mutations can be made in the intermolecular pseudoknots that bind adjacent pRNA subunits, to generate hetero-oligomeric rings of pRNA. It was determined that pRNA mutants that cannot form dimers also cannot support packaging; likewise, pRNA incapable of trimerization also failed to support packaging, indicating that the relevant oligomeric state is a multiple of two and three. These and related experiments using analytical centrifugation suggest that the pRNA assembles as a hexamer. A recent singlemolecule fluorescence experiment provided additional evidence in support of the hexamer model.16 Stalled complexes containing Cy3-tagged pRNA were examined in total internal reflection fluorescence (TIRF) microscopy and identified as fluorescent spots; the number of RNA molecules at a spot was determined by counting the number of photobleaching steps. When the finite labeling efficiency was considered, the data were best fit by a binomial model in which six pRNA molecules are bound to a prohead. In contrast, the most recent and advanced cryo-EM studies of proheads reveal five pRNA subunits symmetrically arranged in a ring, bound to the prohead.49 Imaging of highly symmetric particles such as viruses can contain artifacts due to the computational imposition of symmetry during the averaging process. However, new computational and biochemical techniques introduced to control for these artifacts have confirmed that the pRNA density exhibits fivefold symmetry.15,36,54,55 One potential solution to the discrepancy between these experimental approaches is that six pRNA molecules are present, but only five are symmetrically organized in the packaging complex. The most recent singlemolecule fluorescence experiments provide circumstantial evidence of this hypothesis: it was found that pRNA incapable of supporting packaging nonetheless can bind to proheads with weak affinity, yielding one or zero photobleaching steps,46 thus suggesting a systematic overestimate of the pRNA number. Nonspecific binding of one pRNA to the prohead is detectable with fluorescence but probably not with averaged cryo-EM reconstruction. Moreover, if an additional pRNA molecule is nonspecifically bound to a prohead, as we suggest, it may not be functionally relevant and may not contribute to the organization of the ATPase oligomer.

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4.22.2.4

Portal or Connector

crystallized as a tridecamer, which differs from the known dodecameric state of the prohead-bound portal. Cryo-electron microscopy and subsequent modeling of the high-resolution structure predicts that in the prohead, the tunnel inner diameter is as little as 18 A˚ – too small for DNA to pass. The tunnel is constricted by the ‘tunnel loop,’ a flexible but structured region that contains a conserved acidic residue. In addition to its flexibility, the tunnel loop may be mobile because it is at the end of an alpha-helical lever. Based on these two facts, it was suggested that the loops can move in a coordinated manner during packaging, hence permitting DNA passage and perhaps actively driving the DNA.63 Even allowing for significant tunnel loop flexibility, the small size of the opening seems to be at odds with recent observations that f29 can package both loops65 and bulges,66 as can l,67 and possibly also T4.68 Since packaging of those DNA structures may require threading four ssDNA strands through the channel, it seems likely that the connector as a whole must be capable of considerable conformational flexibility. We will discuss the functional implications of these experiments in the next section. Once packaging is complete, the DNA translocation complex dissociates from the portal, suggesting that the portal undergoes a conformational change such that the channel closes and the ATPase can no longer bind; the naked portal subsequently binds to the tail complex. Upon binding of tail proteins, the P22 portal undergoes a further conformational change which is hypothesized to stabilize the DNA within the capsid, and to recruit additional virulence factors required for

The DNA enters the capsid via a channel called either the portal or the connector. Both names reflect the multifaceted role of this protein oligomer: it is a passageway, structural platform,49 and signal transducer,56 and it has been proposed to interact directly with the DNA during translocation,56,57 among other functions (reviewed in Refs. 58, 59). Across all known packaging dsDNA bacteriophage families, the portal is a dodecameric ring located at a unique fivefold vertex of the icosahedral capsid.49,60–63 The crystal structure of the SPP1 portal63 is the most complete structure available, and the crystal structure of the f29 connector has revealed nearly identical folds despite little sequence similarity.49,60 The overall shape of the connector is a funnel, with a wide aperture nested within the capsid and a narrow end that extends outside (Figure 4). At the wide end are SH3 domains likely responsible for oligomerization and contact with the capsid (the wing). Also within the capsid, but closer to the central pore, is the crown domain thought to contact DNA when packaging is complete and, in some phages, aid in the termination of packaging. The central domain of the connector consists of multiple alpha-helices that run along its length (the stem), forming a near-continuous barrel. The external domain of the connector, called the clip domain, appears to be the assembly point for the rest of the packaging motor.36,64 At its narrowest point, the inner diameter of the crystal structure of SPP1 is 27 A˚ (A˚ngstroms). However, the portal

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Figure 4 Structure and conformational changes of portal/connector. (a) Crystal structure of the SPP1 portal. The free portal crystallized as a 13-mer but is known to be a 12-mer in the capsid. (b) Domains of the portal. The wing domain (red) contacts the capsid; the crown domain (green) faces the capsid interior; the stem domain (cyan) leads to the clip domain (magenta) which faces the ATPase. The tunnel loops (black) are proposed to contact DNA at the interior of the tunnel, and they may be coupled to changes in the kinking of the helix at the intersection of the tunnel loop and wing domains. (c) Conformational change associated with completion of packaging in P22 phage. At the top is a cryo-EM image reconstruction of the free portal; below is a reconstruction of the portal component of a fully assembled virion. A ring of DNA (orange) can be seen surrounding the portal crown domain. Parts (a) and (b) Adapted from Lebedev, A. A.; Krause, M. H.; Isidro, A. L.; Vagin, A. A.; Orlova, E. V.; Turner, J.; Dodson, E. J.; Tavares, P.; Antson, A. A. Structural framework for DNA translocation via the viral portal protein. EMBO J. 2007, 26(7), 1984–1994, adapted by permission from Macmillan Publishers Ltd. Part (c) reprinted from Lander, G. C.; Tang, L.; Casjens, S. R.; Gilcrease, E. B.; Prevelige, P.; Poliakov, A.; Potter, C. S.; Carragher, B.; Johnson, J. E. The structure of an infectious P22 virion shows the signal for headful DNA packaging. Science 2006, 312(5781), 1791–1795, with permission from AAAS. Courtesy of G. Lander and J. Johnson.

Viral DNA Packaging Motors injection.69 Thus, the portal is intimately involved in all stages of viral assembly, packaging, and infection.70

4.22.2.5

Capsid

After passing through the portal, the DNA reaches its destination, the capsid. As it defines the volume into which the genome must be compressed, the capsid is an important determinant of packaging behavior. The dsDNA phages share a basic icosahedral shape, typically about 50 nm across, that can vary in radius and length depending on species (reviewed in Refs. 71, 72). Located at one fivefold vertex of the head is the dodecameric connector (Figure 2) through which DNA passes during packaging. Between the initial capsid assembly and the completion of packaging, the capsid undergoes significant structural rearrangement involving processes such as proteolysis,73 crosslinking,74 departure of the scaffold,75 and volume expansion,73 with different bacteriophages displaying different combinations of these maturation events. Some of these rearrangements occur during and are, perhaps, triggered by packaging itself. For example, the scaffold proteins of f29 exit during packaging,76 and many phages undergo expansion during packaging.43,77

4.22.3

How is Packaging Initiated?

The initiation of packaging differs slightly between different tailed bacteriophages, with some phages recognizing a particular sequence and others recognizing a terminal marker protein. In this section, we will outline the current understanding of the molecular mechanism of initiation in these two systems.

4.22.3.1

Initiation via Terminal Protein

The terminal protein of the f29-like phages plays an important role not only in packaging, but also in genome replication (reviewed in Refs. 5, 22, 78, 79). Replication of the genome by the viral polymerase requires a protein primer, yielding unitlength linear viral replicons covalently bound to the protein at both 50 ends (Figure 1(a)). Terminal protein bound to at least one end of the DNA segment is required for packaging in vitro although the mechanism by which it facilitates packaging is not yet fully understood. Terminal protein induces a loop topology into otherwise unstructured DNA, and those loops or lariats can be supercoiled by the packaging machinery;80 it has been proposed that supercoiling is involved in packaging initiation.47,80,81 Single-molecule experiments have suggested that such loops do form in solution and can be packaged.65 Interestingly, in optical tweezers, DNA that is not terminally labeled can be packaged, albeit with low efficiency.65 The dynamics of packaging such DNA are indistinguishable from those of packaging natural phage DNA, signaling that the effect of this protein lies not in processive translocation, but in some other part of the packaging process. Specifically, the fact that removal of the terminal protein reduces but does not abolish initiation events in singulo suggests that its role is to assist the search phase of initiation. After initiation, the left-

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end protein, being covalently linked to the DNA being packaged, is likely to enter the capsid. The terminal protein at the right end of the DNA appears to become wedged in the tail itself after the conclusion of packaging,82 suggesting that it helps to stabilize the highly pressurized packaged particle.

4.22.3.2

Initiation via Terminase Small Subunit

For bacteriophages without a terminal protein, initiation of packaging begins with binding of the terminase small subunit to the virally replicated DNA, in most cases to a specific cos or pac sequence (Figure 1(b)) (reviewed in Refs. 4, 7). Because DNA replication in these systems produces a long DNA polymer of concatenated genomes, multiple binding sites may be present. The small subunit recruits the terminase large subunit to cleave the DNA at or near that site. The terminase holoenzyme must subsequently bind to a prohead to begin packaging from the freshly exposed DNA end. Upon completion of packaging, the DNA is cleaved again, and the terminase–DNA complex diffuses away to find another empty capsid. The exact location of termination may be sequencespecific for some phages (reviewed in Ref. 43) or variable for others (reviewed in Refs. 83, 84) like those utilizing the ‘headful’ mechanism to be described below. Sequence-specific cleavage, such as cleavage of the cos site by l, is controlled by the terminase small subunit85 but executed by the large subunit.86 When the small subunit oligomerizes into a ring, the specific DNA-binding domains are located on the outside of the ring,24 possibly causing the DNA to loop as it is threaded through the ring to the terminase large subunit’s nuclease domains.25 Interestingly, the small subunit is dispensable for packaging in vitro and in singulo by T4.42,87 In fact, in the absence of the small subunit, terminase large subunit packaging and nuclease activity are both high and nonspecific in vitro,42 suggesting that the small subunit acts as an allosteric regulator of large subunit function. When the small subunit oligomer is specifically bound to a cos or pac site on the phage genome, it may deregulate nuclease activity, hence kinetically promoting cleavage over translocation. At other DNA sequences, the small subunit may assume a conformation that suppresses cleavage, thereby favoring translocation.

4.22.4

How is the DNA Translocated?

After packaging is initiated, the viral genome must be directionally pumped and compressed into the capsid. Of all of the steps in the packaging process, the translocation of DNA by the packaging motor has been, perhaps, the best studied with biophysical methods. This focus is not surprising: double stranded nucleic acid is a particularly, stiff, highly charged molecule, and the fact that the packaging complex compresses this molecule to high densities (typically 300 to 500 mg ml–1) is one of the most remarkable features of the genome packaging process. In this section, we discuss several basic features of the DNA translocation process: what are the forces involved in this process, how is chemical energy converted into mechanical energy, how does the packaging complex engage the DNA and apply force to it, and finally what are the core

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structural elements responsible for engaging and performing work on the DNA.

4.22.4.1

Single-Molecule Measurements of Velocity of Packaging

One of the basic features of the packaging process is the rate at which DNA is pumped into the capsid (i.e., the velocity of packaging). Time-resolved nuclease protection assays provide estimates of the rate at which DNA is packaged, convolved with other processes;88 however, more direct measurements of these velocities have recently come from optical tweezers assays (reviewed in Refs. 89, 90), in which the length of DNA packaged by a single complex is monitored directly (Figure 5(a)). Optical tweezers are a powerful tool for measuring forces at the picoNewton (pN; 10–12 N) scale and measuring displacements at the nanometer scale, which are the regimes within which packaging viruses operate. An optical tweezers consists of a laser beam focused to a small spot. A micron-sized dielectric particle (usually a polystyrene sphere) is attracted to the laser spot with a force that is proportional to the distance of the particle from the spot; in this way, the laser acts as a trap that can be used to apply specific forces to the particle (Figure 5(a)). In addition, the laser light that is scattered by the particle can be collected and used to infer both the force on the particle and the displacement of the particle away from the center of the laser with sub-nanometer accuracy – far better than conventional diffraction-limited optical microscopy. In order to observe packaging with optical tweezers, the complex, consisting of the prohead, packaging motor, and DNA, must be attached to two micron-sized beads (Figure 5(a)). The DNA can be attached to a bead by modifying the distal DNA end with biotin, a small molecule which can be tightly bound by streptavidin, a protein which is coated onto the surface of a polystyrene bead. The other end of the packaging complex – the prohead – is affixed to another bead via antibodies to the phage capsid protein. In the tweezers, the complex is stretched between the two beads, allowing the tension and extension of the DNA to be monitored in real time as the DNA is packaged. f29, the first bacteriophage to be studied with optical tweezers,10 has a maximum packaging velocity at room temperature of B120 bp s–1 (Figure 5(b)). At this speed, the full 20-kbp genome would be packed in less than 4 min, significantly less time than is apparent in bulk in vitro experiments (cf. Ref. 91). However, the velocity decreases as more DNA is packaged (Figure 5(c)). In contrast, T4, which has a 171-kbp genome, occasionally reaches speeds as high as 2000 bp s–1 but averages around 700 bp s–1;87 l, with an intermediate-sized genome, packages at about 600 bp s–1.92 The average packaging velocity in these systems scales roughly with genome length, inviting speculation as to a biological rationale. This time frame may allow the packaging process to avoid outpacing the production and assembly of packagingcompetent proheads. Another difference between these three phage systems is the variation in the packaging velocity of single complexes (Figure 5(b)). f29 packages in a very uniform manner while T4’s velocity fluctuates significantly from

moment to moment. This observation suggests that the T4 packaging motor may have several distinct functional states with different packaging velocities and which can freely interconvert – a phenomenon known as dynamic disorder.

4.22.4.1.1

Packaging velocity depends on force – Thermodynamics of packaging

The ability to apply defined forces with an optical tweezers while measuring packaging velocities allows the force dependence of translocation to be measured. The application of force allows the energy landscape to be controllably modified due to an additional term, FDx, which results from the mechanical work required to move a distance Dx against a force F. The dependence of packaging on force is thus a singularly powerful quantity because it contains information about the energy landscape along which packaging transpires (Figure 5(d)). The force generating properties of a molecular motor can be characterized with two basic numbers: the stall force and the distance to the mechanical transition state.93 Optical tweezers studies have identified the force at which the packaging process is effectively stalled for f29, T4, and l.10,87,92 In all three cases, the measured stall force is on the order of 60 pN, placing these motors among the strongest known (Figure 6(a)). Moreover, DNA undergoes a structural transition at B67 pN (reviewed in Ref. 94); thus, the packaging motors are essentially as powerful as permitted by their substrate. Thermodynamically, the stall force is the force at which the mechanical work required to package DNA against an applied load is exactly equal to the energy derived from nucleotide hydrolysis. Applying forces beyond the stall force should, in theory, drive the motor in reverse, unpackaging DNA and generating NTPs from NDPs and phosphates. However, it is likely that the experimentally measured stall forces are actually underestimates of the thermodynamic stall forces. First, there is experimental and theoretical evidence that the optically applied force is not the only force resisting packaging. As the genome is compressed within the capsid, it becomes pressurized, reaching a final pressure as great as several tens of atmospheres.10,95 When the resisting force of this backpressure is included with the optically applied force, the estimate of the stall force in f29 jumps to 110 pN.96 In addition, none of the probed molecular motors have been observed to run backwards when larger forces are applied; thus, the measured ‘stall’ forces cannot be truly called thermodynamic stall forces. Instead, what is measured is more likely an operational stall force and the slow down with force arises from a force-dependent entry into off-pathway, paused states. Similar behavior is believed to dominate the force dependence of both eukaryotic and prokaryotic RNA polymerases.97–100 While the stall force is the force at which the packaging process is halted, the distance to mechanical transition state determines the slope at which packaging slows with force. Intuitively, the distance to mechanical transition state can be crudely thought of as the distance through which the DNA must move before it is committed to being packaged. Because the packaging motor must work against an opposing force, a large distance to mechanical transition state means that the motor (or thermal bath) must expend more energy to trigger translocation than if the distance were small. As a

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Figure 5 Optical tweezers observations of phage packaging. (a) Experimental set-up. The capsid is immobilized onto a micron-sized polystyrene bead via antibodies while the far end of the DNA is attached to another bead via a biotin-streptavidin linkage. Each bead is held in a focused laser spot, allowing measurement and application of forces and positions. Some experiments use only one optical trap, with the other bead immobilized by suction onto a micropipette. (b) The length of the DNA between the beads decreases as a function of time due to packaging. At the beginning of packaging, when the capsid is empty, l (red) and f29 (green) package DNA at a constant velocity. T4 (black), in contrast, displays dynamic disorder. (c) The velocity of packaging by f29 (top) drops as more DNA is packaged, whereas the velocity of packaging by l (bottom) shows more complex behavior attributed to structural changes in the capsid. (d) Hypothetical example of how force modifies the energy landscape of a molecular motor. In this example, the motor takes steps (as indicated by the distance between minima in the potential Dx). The horizontal distance between a minimum and the nearest maximum is the distance to the transition state Dxt, and the energy difference between minimum and maximum is the activation energy of a step DGt. When a resisting force, or load, is applied to the motor, the motor must perform additional mechanical work FDx to move against the force. Likewise, the height of the barrier to stepping is increased by a mechanical term FDxt. (a) Data generously provided by G. Chistol, D. Smith, D. Fuller, and J. Tsay. Data in parts (b) and (c) reprinted from Fuller, D. N.; Raymer, D. M.; Rickgauer, J. P.; Robertson, R. M.; Catalano, C. E.; Anderson, D. L.; Grimes, S.; Smith, D. E. Measurements of single DNA molecule packaging dynamics in bacteriophage l reveal high forces, high motor processivity, and capsid transformations. J. Mol. Biol. 2007, 373 (5), 1113–1122 and Rickgauer, J. P.; Fuller, D. N.; Grimes, S.; Jardine, P. J.; Anderson, D. L.; Smith, D. E. Portal motor velocity and internal force resisting viral DNA packaging in bacteriophage f29. Biophys. J. 2008, 94 (1), 159–167.

consequence, the packaging process will be slowed more drastically with applied force for larger distances to transition state (Figure 5(d)). For f29, a detailed fit of the average rate of DNA packaging as a function of applied force revealed a remarkably small distance to transition state, just B1.1 A˚

(Figure 6(a)).11 T4 shows an even weaker force dependence than f29, suggesting that its distance to transition state is even smaller.87 In contrast, the packaging motor of l displays more complicated behavior. At low forces, l appears to have a larger distance to transition state, B3 A˚; however, at high forces, the

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Figure 6 Force dependence of packaging velocity. (a) Packaging velocity as a function of external force under saturating [ATP], normalized to the value at 5 pN. (b) Michaelis–Menten parameters, Vmax and KM, as a function of force for the packaging motor of f29. (c) The force dependence of the ratio of these two parameters. Data in (a) previously published in Fuller, D. N.; Raymer, D. M.; Kottadiel, V. I.; Rao, V. B.; Smith, D. E. Single phage T4 DNA packaging motors exhibit barge force generation, high velocity, and dynamic variability. Proc. Natl. Acad. Sci. USA 2007, 104, 16868–16873. Copyright by PNAS; Fuller, D. N.; Raymer, D. M.; Rickgauer, J. P.; Robertson, R. M.; Catalano, C. E.; Anderson, D. L.; Grimes, S.; Smith, D. E. Measurements of single DNA molecule packaging dynamics in bacteriophage l reveal high forces, high motor processivity, and capsid transformations. J. Mol. Biol. 2007, 373(5), 1113–1122 and Rickgauer, J. P.; Fuller, D. N.; Grimes, S.; Jardine, P. J.; Anderson, D. L.; Smith, D. E. Portal motor velocity and internal force resisting viral DNA packaging in bacteriophage f29. Biophys. J. 2008, 94(1), 159–167 and provided by D. Smith. Data in (b) and (c) reproduced from Chemla, Y. R.; Aathavan, K.; Michaelis, J.; Grimes, S.; Jardine, P. J.; Anderson, D. L.; Bustamante, C. Mechanism of force generation of a viral DNA packaging motor. Cell 2005, 122(5), 683–692.

packaging velocity of l becomes remarkably insensitive to force.92 One explanation of this is that l moves the DNA in two steps, which have different distances to transition state and, thus, become rate-limiting under different force regimes. However, the initial rapid decrease in packaging velocity with force requires that the more sensitive force-dependent step be initially rate limiting, and a simple linear two-step model does not explain how the less force-sensitive step can overtake the more force-sensitive step. An alternative explanation is that these two force-dependent steps do not occur along the same kinetic pathway, but rather along alternative kinetic routes. In other words, as force is increased, l actually may preferentially utilize a different packaging mechanism.

4.22.4.1.2

Velocity depends on substrate – Identifying mechanical and chemical transitions

In addition to precisely controlling the applied force in the packaging process, the single molecule optical tweezers assay permits careful control over the chemical environment of the packaging motor. By titrating the concentration of NTPs as well as the products of the hydrolysis reaction, NDPs and Pi, the chemical portion of the mechanochemical conversion could be deciphered. Of the three packaging motors studied with optical tweezers, extensive mechanochemical studies have been conducted only with f29.11 Here it was found that the packaging velocity had a simple Michaelis–Menten dependence on the concentration of ATP, with a maximum velocity, Vmax, of B120 bp s–1 and a Km of B30 mM. As increasing force was applied, both the Km and Vmax decreased, yet the ratio of these two quantities remained constant (Figure 6(b), (c)). In addition, it was found that large concentrations of inorganic phosphate had no measurable effect

on velocity under conditions of both low (B5 pN) and high (B40 pN) opposing loads, indicating that the release of phosphate involves a large change in free energy and is effectively an irreversible kinetic transition. These observations greatly restrict possible mechanisms for the mechanochemical conversion in f29. First, force is generated either simultaneously with or immediately after a largely irreversible transition, because translocation is irreversible; thus, the kinetic (chemical) process that occurs concomitantly with force generation must be irreversible. Second, the relationship of Km and Vmax with force turns out to mean that ATP binding – an event during which significant amounts of enthalpic energy are generated via a zippering of hydrogen bonds with the phosphate backbone101 – cannot be the force generation step. Third, data not shown here reveal that ADP binds reversibly to the motor, strongly implying that release of ADP cannot be the force generating step. Finally, the fact that phosphate release is irreversible and appears to involve a large change in free energy reveals it to be the most likely candidate for the chemical process that corresponds to the translocation step.

4.22.4.1.3

The role of sequence motifs in force production

Members of the ASCE clade, including the packaging motors, have a variety of defining sequence motifs including the common Walker A, Walker B, catalytic glutamate, and arginine finger,33,102,103 and may have additional features such as the Q-motif and C-motif.3,104 For a general schematic of the positioning of these motifs in the context of secondary structure, see Figure 3; for more detailed information, many excellent reviews are available.3,102,103 The Walker A motif, which is identical to the P-loop common to many other

Viral DNA Packaging Motors

NTPases, is a flexible loop that hydrogen bonds to the phosphate groups in the ATP. Positioning of the ATP is also assisted by a motif sometimes called the Q-motif which is thought to directly bind the adenine and is located just upstream of the Walker A. Also spatially nearby is the Walker B motif (but downstream, after beta strand 2), which coordinates a magnesium ion to orient the gamma-phosphate bond. The catalytic glutamate residue, often but not exclusively located one residue downstream of the Walker B motif, positions and activates a water molecule for nucleophilic attack on the gamma phosphate bond. Protruding into the nucleotidebinding pocket, usually from an adjacent subunit, is an arginine finger which couples ATP binding and hydrolysis to larger conformational motions, and is likely to stimulate hydrolysis as well. A similar coupling role is played by two downstream motifs which have been identified in different proteins: the sensor motif, identified in the AAA þ family, and the C-motif, identified in helicases (where it is known as motif III) and in the terminase packaging motors. In recent single-molecule studies, Tsay and coworkers have begun to determine the role of these additional motifs in the generation of force and processivity of the l packaging motor.105,106 By using an optical tweezers assay to follow packaging of mutant l packaging motors, these authors found that mutations within or near the Walker A and B motifs decreased the packaging velocity without affecting the force sensitivity of packaging. These results are consistent with the ATP-binding and hydrolysis role for the Walker A and B motifs and further suggest that while these motifs are necessary to transduce the energy for DNA translocation, they do not directly determine the magnitude of force generated by the motor. It was also found that a mutation in the Q-motif similarly decreased the average packaging velocity without affecting ATP binding; however, in contrast to the Walker A and B mutations, this mutation also dramatically increased the force sensitivity of packaging, and reduced processivity.107 This result provides direct support for the emerging picture that the Q-motif, which is known to be directly involved in binding the adenine group of ATP,108 is also responsible for coupling conformational changes in the ATP binding pocket into motion of the substrate.109 Mutations downstream of the ATP-binding motifs also have effects on packaging. A point mutation to the helix immediately following the Walker B motif greatly reduced velocity,106 as did the homologous mutation in the FtsK-clade dsDNA translocase SpoIIIE,110 an effect attributed to a slight structural change. Finally, a mutation in the C-motif resulted in a decrease in both the velocity and processivity of the motor, suggesting a more direct role in the interaction between the motor and the DNA than suggested from biochemical work on related motors.104 Moreover, second-site pseudorevertants of the C-motif mutation (second mutations that restore wild-type behavior) were found to exist scattered throughout the ATPase. The consensus interpretation of these data is that subtle tertiary interactions in the protein can have a dramatic effect on catalysis. It is therefore likely that changes outside of functional motifs will have phenotypic effects, making it difficult to disentangle the specific roles of various residues. Determining the exact functions of the residues in the packaging ATPases will require careful examination, using

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single-molecule techniques, of the base pair-scale dynamics of packaging by a wide variety of mutants.

4.22.4.2

High-Resolution Observations of Packaging

Taken together, these measurements provide strong constraints on the mechanochemical conversion process of a single subunit; however, the packaging motors are all multimeric rings of identical subunits, and these studies provide little information concerning the way in which the subunits in the packaging motor coordinate their activities. High resolution optical tweezers, capable of following the discrete increments of DNA packaged each cycle of the packaging motor, have recently been developed to address this question in f29.12 These optical tweezers experiments were able to resolve not just single molecules, but single turnovers of the enzymatic cycle.

4.22.4.2.1

Base-pair-resolution measurements reveal step size and coordination of stepping

At low forces, it was observed that DNA was packaged in increments of 10.070.2 bp, independent of the concentration of ATP.12 Closer inspection of these packaging traces revealed that the 10 bp packaging events were actually bursts of multiple smaller steps. By applying large opposing forces to the packaging motor, these bursts of steps could be slowed sufficiently to measure the size of the steps within each burst. Remarkably, it was found that the f29 motor packages the DNA in a non-integer number of base pairs, 2.4 7 0.1. These observations strongly suggest that each full mechanochemical cycle of the motor is composed of two phases: (i) a dwell phase in which at least four of the five subunits bind ATP sequentially, each delaying the utilization of this molecule until the ring is loaded and (ii) then a burst phase in which these subunits cooperatively generate a series of four 2.5 bp steps in quick succession (Figure 7(a), (b)).

4.22.4.2.2

High-temporal-resolution measurements reveal coordination of ATP binding

Further support for a segregated burst-dwell model came from a detailed study of the pauses between the 10 bp packaging events under low opposing load. Because the scale of energy splittings between different states of a molecular motor is similar to that of the surrounding thermal bath, the time to complete a single mechanochemical cycle is subject to large fluctuations. It was found that the time for completing a cycle was most variable under conditions of limiting or saturating ATP, and least variable at intermediate concentrations of ATP. More subtly, it turns out that the degree to which these pause durations are variable places limits on the number of kinetic transitions that are rate-limiting under specific ATP concentrations – a result first proved by Aldous and Shepp in the context of queuing theory.111 This limit is captured by a quantity that we have termed nmin – the inverse of the squared coefficient of variation.12,112,113 A fit of nmin versus [ATP] to a general expression – akin to the Michaelis–Menten equation – revealed that the pause before each 10-bp event must involve the binding of at least two ATP molecules before each 10-bp burst and at least four additional non-ATP binding events. The fact that four steps compose each 10-bp burst strongly suggests

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Burst phase Figure 7 Intersubunit coordination in the packaging motor of f29. (a) Schematic diagram of the two-phase cycle superimposed on an actual packaging trace. During the dwell phase, the motor loads four ATPs while holding the DNA at constant length. During the burst phase, the motor uses these ATPs to package the DNA in four 2.5-bp steps. (b) Sample packaging traces under high opposing loads. The fast 2.5-bp steps are not observable under low forces (red), but under high forces they are clear (black) . (c) Schematic picture of the full mechanochemical cycle of the packaging motor. During the dwell phase, a single subunit is capable of binding ATP (green) and once it does (red), it activates the adjacent subunit. Once fully loaded, the motor enters the burst phase where, triggered by Pi release, it takes a series of 2.5-bp steps. Blue subunits correspond to subunits that may contain product ADP and the purple subunit signifies the fifth, distinct subunit. This ‘special’ subunit may change each cycle. A careful analysis of fluctuations in the dwell phase duration reveals that each of the four subunits must have a catalytic efficiency (ratio of kcat to KM) that must vary by roughly a factor of four from the previous subunit. While depicted as increasing here, the data cannot uniquely determine whether the catalytic efficiencies decrease or increase around the ring. Adapted from Moffitt, J. R.; Chemla, Y. R.; Aathavan, K.; Grimes, S.; Jardine, P. J.; Anderson, D. L.; Bustamante, C. Intersubunit coordination in a homomeric ring ATPase. Nature 2009, 457(7228), 446–450. Copyright by Nature.

that four ATP molecules bind to the ring in each full mechanochemical cycle. The fluctuation analysis is not inconsistent with the binding of four ATPs as nmin values only provide lower limits on the number of kinetic events. However, reconciling the binding of four ATPs with a measured value of B2 requires that the chemically identical subunits do not bind ATP with the same rates. The catalytic efficiency (kcat/ KM) provides a measure of the rate at which substrates are bound and committed to the cycle; and the fluctuation analysis reveals that, on average, each subunit must increase (or decrease) in catalytic efficiency by a factor of B4 with respect to the previous subunit.113 Such a kinetic asymmetry in chemically identical subunits suggests that extensive and subtle communication must occur between the subunits in the packaging motor. Recent co-crystal structures of distantly related ring motors and their nucleic acid substrate reveal the type of structural asymmetries between subunits that would be required to produce this behavior in the packaging motor of f29.103,114 The full mechanochemical model for the packaging motor of f29 is summarized in Figure 7(c). In this way, high-resolution measurements have revealed three essential asymmetries of this ring-shaped ATPase. First is

a mechanical asymmetry – despite having five subunits, the motor moves the DNA in four consecutive increments before dwelling to bind ATP. Then, the motor binds either four or five ATPs, with each ATP being bound at a different rate – a kinetic asymmetry. And finally, individual ATP binding events and DNA stepping events are strictly separated in time, taking place in an ordered manner. Such profound asymmetries between subunits reveal that the subunits are not so identical as they appear. As seen in Figure 7(c), there must be one ‘special’ subunit that does not translocate DNA during one 10bp burst, and may not bind ATP at all. These asymmetries can be seen within an individual cycle. How the fivefold symmetry of the motor is broken, and whether it is broken differently from one cycle to the next, remains unknown.

4.22.4.2.3

Thermodynamic efficiency

With a measured step size for the f29 packaging motor, it is possible to calculate the thermodynamic efficiency of this motor. This efficiency is a commonly used number as it provides a simple way to compare these microscopic motors to their macroscopic counterparts; however, care should be taken in considering efficiency on the molecular level. Under normal

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4.22.4.3

Observations of Packaging on Modified Substrates

DNA displays a chemically diverse molecular surface. The major and minor grooves display chemical moieties capable of forming hydrogen bonds and of being polarized while the phosphate backbones can make strong ionic interactions. A crucial part of understanding the mechanism of the packaging motors is determining how they engage this remarkable surface; how force is transduced to the DNA substrate; and how, in turn, interactions with this substrate regulate the mechanochemical cycle. Several recent experimental efforts, varying from single-molecule to crystallographic, have provided some surprising answers to these questions.

4.22.4.3.1

Backbone phosphates provide processivity and regulate the chemical cycle

To address these issues in the packaging motor of f29, Aathavan et al.66 challenged the packaging motor with a DNA molecule that had been locally modified and monitored packaging as the complex encountered the modification with an optical tweezers assay. To probe the importance of ionic contacts with the charged phosphate backbone, a neutrally charged, structurally identical mimic of DNA was tested. The packaging motor successfully packaged neutralized DNA of various lengths from 5 to 30 bp with relatively high efficiency (greater than 10%). By reintroducing charge to each strand selectively, it was found that the most important phosphate contacts are made with adjacent phosphates on the 50 –30 strand, in the direction of packaging, every 10 bp. At the base pair scale, it was observed that the absence of a phosphate charge on that strand results in a significant increase in the frequency with which the motor loses attachment with the DNA, revealing that phosphate contacts are necessary for high processivity. In addition, it was found that the rate at which the motor attempts to package the DNA decreased 10-fold in the absence of the phosphate charge. This reduced attempt

rate was largely due to a single slow kinetic event. Thus, in the f29 packaging motor, contacts with phosphate charges on the 50 –30 strand every 10 bp provide the motor with its high processivity and appear to play a role in the regulation of the chemical cycle.

4.22.4.3.2

Non-nucleic-acid-specific contacts drive translocation

To probe contacts with additional chemical moieties, the f29 motor was challenged with a wide range of chemically modified DNA strands, including abasic DNA, single stranded gaps, unpaired bulges, and even a chemical linker with no resemblance to DNA. Remarkably, the motor was capable of packaging all of these modifications with relatively high efficiency. Since the complement of missing elements packaged by the motor include all of the chemical moieties of DNA – phosphates, sugars, and bases – the only conclusion is that none of the individual chemical features of DNA are absolutely required for packaging. By performing a multivariate logistic regression – a standard statistical method that extracts the relevance of one variable while controlling for changes in others – Aathavan et al. were able to quantify the ‘importance’ of each of the different chemical moieties of DNA.66 The results can be summarized in a heat-map of the interactions between DNA and the motor (Figure 8). The picture that emerges then is that important phosphate contacts are made by the motor every 10 bp on the 50 to 30 strand oriented in the direction of packaging. In between these ionic contacts, the 0.16 Translocation burst

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physiological conditions, ATP hydrolysis releases roughly 100 pN nm (10010–21 J) of energy (for comparison, the energy due to the thermal bath is about 4.1 pN nm, or 0.59 kcal mol–1). When there is no applied load to the packaging motor or internal pressure on the genome, all of this energy must go into heat, in other words, the motor is 0% efficient. When larger forces are applied, then some of this energy can be converted into mechanical work, and the motor takes on a finite efficiency. The maximum thermodynamic efficiency of the packaging motor is reached at the stall force, where paradoxically, the motor does not package DNA at a finite rate. With a step size of 2.5 bp and an estimated stall force of B110 pN, the packaging motor is capable of generating B90 pN nm of mechanical work. Since ATP can provide B100 pN nm, depending on environmental conditions, the packaging motor has an efficiency of almost 90%, comparable to the most efficient known molecular motor, F1ATPase/F0-ATP synthase.93,115 While exact measurements of the step size have not been made for other packaging motors, bulk estimates for the step size are of the same order B2 bp/ ATP, suggesting that high thermodynamic efficiency is a common feature of the packaging motors.

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0.00 Figure 8 Heat map of importance of interactions between DNA and the packaging motor of f29. Contact importance is defined as the reciprocal of the number of base pairs of a given chemical moiety (phosphate charge or sugars/bases) that must be removed to decrease the probability of packaging this lesion to 50%. The most important contacts are with adjacent phosphates on the 50 –30 strand in the direction of packaging (down in this image). These contacts are likely made during the dwell phase. During the burst phase, contacts are made with all other chemical moieties on both strands. Reproduced from Aathavan, K.; Politzer, A. T.; Kaplan, A.; Moffitt, J. R.; Chemla, Y. R.; Grimes, S.; Jardine, P. J.; Anderson, D. L.; Bustamante, C. Substrate interactions and promiscuity in a viral DNA packaging motor. Nature 2009, 461 (7264), 669–674. Copyright by Nature.

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motor appears to make non-specific, steric contacts every 2.5 bp. Along these lines, mutational studies of the related ASCE motor, simian virus 40 hexameric helicase, provide some support for the necessity of non-specific, perhaps steric contacts.116 It is important to note that while the units of ‘contact importance’ are quantitative and formally defined66 (as the inverse of the number of base pairs of instances of the perturbation that are required to reduce the probability of successful traversal by 50%), these units are not easily interpreted in terms of interaction energies. However, such measures of dynamic importance provide important complements to the estimates of interaction energies extracted from crystallographic studies, and it will be interesting to see how well these measures agree once co-crystal structures of packaging motors with their nucleic acid substrates are available. This work is supplemented by previous DNA modification studies done with f29117 and T4118 though it does conflict somewhat with the conclusions drawn from those studies. In both cases, it was found that modifications to the DNA greatly disrupt packaging. In the case of f29, Moll and Guo117 found that large stretches of single-stranded gaps could not be packaged, though nicks could be. These results can be reconciled with the results of Aathavan et al. since these authors found that increasing the length of DNA modification significantly decreased the probability of successful translocation and the gaps used by Moll and Guo were likely quite large. In the case of the T4 studies, Oram et al.118 used short oligos as the substrate for the in vitro packaging system; thus, it is possible that discrepancies arise due to the effect of modifications on packaging initiation – not translocation. Support for generality of the strong ionic contacts observed in the burst phase of f29 is provided from a variety of crystallographic sources, including the packaging motor of T428 and other related ASCE motors such as Rho helicase119 and the bovine papillomavirus E1 helicase.120 However, crystallographic studies give little support for the less specific, transient contacts suggested for f29. This discrepancy may be explained by selection bias for stronger, long-lived contacts in the crystallization process; it may well be that evidence of these transient contacts can only be gleaned from dynamic measurements.

4.22.4.3.3

Motor–DNA interaction changes during the cycle

Taking all the facts presented in this section together with knowledge of the intersubunit coordination in f29 discussed earlier, a picture of how the motor–DNA interactions change throughout the cycle emerges. During the dwell phase of the cycle, as the motor loads four ATPs, phosphate contacts on the 50 –30 strand provide the necessary strong contacts to keep the motor bound to the DNA against opposing loads. The fact that removing these contacts dramatically changes the chemical cycle suggests that there is a single chemical transition during this dwell phase which is strongly dependent on the proper positioning of the DNA. This kinetic step might serve as a mechanochemical checkpoint, properly synchronizing the completion of the packaging from the previous cycle with the loading of nucleotides for the next cycle. Since neutral stretches of DNA shorter than 10 bp were readily packaged, phosphate contacts are not nearly as important during the

burst phase, when the DNA is packaged in four 2.5 bp steps, as during the dwell phase. During this phase, it is likely that more transient, non-nucleic-acid-specific contacts are what actually drive packaging. While such promiscuous contacts present a significant challenge to the prevailing view of specific motor–DNA contacts from an energetic perspective, they may also provide one solution to a basic problem. During active packaging, strong contacts are needed to transduce large forces, but these contacts must also be short-lived since quick packaging requires them to break a few milliseconds after they are made. Non-specific, potentially steric contacts may satisfy this requirement. Similar periodic contacts have been inferred from nucleotide-analog-interference studies with the related Rho translocase.121

4.22.4.3.4

The origin of the 2.5-bp step

Finally, the modified-substrate studies of Aathavan et al.66 provide one interesting solution to the mysterious 2.5-bp step size revealed from high-resolution optical tweezers studies of f29. A step size that is a non-integer number of base pairs raises clear questions concerning the interaction between the motor and the DNA since DNA repeats chemically every base pair. The use of non-specific contacts may be the origin of the non-integer step size. During the burst phase, the motor may not need specific chemical moieties to drive packaging, allowing the step size to be set not by the chemical periodicity of the DNA, but by the distance moved by internal conformational changes in the packaging motor. However, as indicated by the high resolution packaging data, the packaging process is not completely decoupled from the chemical periodicity of the DNA – every four steps must add to 10 bp. Specific phosphate contacts during the dwell phase may reconcile this aspect of the process, rectifying any mismatches with the chemical periodicity that remain at the end of each burst phase. However, it is worth noting that there are several ways by which a multimeric motor can generate a non-integer step size without actually contacting the DNA at non-integer positions. For example, if the motor is not a flat planar ring, then the observed step size would correspond to the distance between contacts on the DNA plus any difference in the position of adjacent subunits with respect to the average plane of the motor. One may imagine that the motor forms a shallow helix, cracking the ring in one position and shifting each subunit along the contour length of the DNA by some small amount. If this shift were just B1.7 A˚, this ½ bp offset would allow each subunit to make a 2.5-bp step, yet specifically contact the DNA 2 or 3 bp away. While the DNA modification studies rule out some models by which a non-integer step size can be generated, the origin of this surprising step size has not yet been fully addressed and remains an exciting avenue for future research.

4.22.4.4

Defining the Mechanistic Role of the Different Components

We have identified the features of DNA that are important for packaging; however, this is only half the picture of the motor–DNA interaction. To fully understand how the motor

Viral DNA Packaging Motors

engages and moves the substrate, we must also consider the contribution of the motor – that is, what residues and secondary structures directly contact the DNA, and how they interact to generate force. Because packaging requires the involvement of all the components of the complex – the capsid, connector, and ATPase – the protein component responsible for performing work on the DNA as part of the mechanical step has not been conclusively identified. The ATPase is responsible for ATP binding and hydrolysis; it may directly drive the DNA, or it may induce conformational changes in the connector/portal which in turn move the substrate. In this section, we discuss the two predominant candidates for the identity of the DNA-engaging part of the motor – the connector/portal and the ATPase – and argue that the ATPase is likely to perform the task of DNA translocation.

4.22.4.4.1

The ATPase may engage and drive the DNA

The dsDNA packaging bacteriophages universally contain a protein of the ASCE division of ATPases. The ATPases fall within a larger class, comprising 5–10% of the predicted gene products (including G-proteins, some kinases, and some cytoskeletal proteins, among others) among all genomes, termed the P-loop NTPases because they contain a conserved phosphate-binding loop.33 The ASCE division itself is highly diverse, including proteins that transport a wide variety of nucleic acid, protein, and small-molecule substrates.32,33 Sensitive bioinformatic studies have found that about 15% of the packaging phages, including f29, possess an ATPase of the FtsK/HerA clade of dsDNA translocases (Figure 3).20 FtsK, the clade prototype, is a homohexameric ring motor responsible for chromosomal segregation during bacterial cell division,122 so it seems likely that these phage ATPases perform a similar DNA pumping function in viral replication. In contrast, the terminases – found in the other well-studied phages such as l, T4, and SPP1 – are not so easily classified: although they are clearly ASCE proteins, they lack some of the secondary structure features that differentiate the other well-characterized ASCE clades and thus appear to be an independent lineage within the ‘Strand 2 Insert’ division of the ASCE family (Figure 3).20 The terminases do contain the Q motif and C motif, which are thought to help couple ATP hydrolysis to translocation in the monomeric helicases but are not found in RecA or F1-ATPase.104–106,123 Other DNA-binding lineages within the Strand 2 Insert division, such as RecA and the monomeric helicases, do not form nucleotide-stabilized symmetric rings despite binding nucleotide at the subunit interfaces. Hence, cladistics provides only limited insight into the function, structure, and coordination of the terminase large subunits. There is no direct evidence that purified, isolated ATPase induces translocation of DNA. All terminases exhibit ATPase activity that is stimulated by DNA, although hydrolysis is maximized in the presence of all packaging complex components.42,56,104,124,125 DNA binding has been documented in many packaging ATPases, although this could be mediated through the endonuclease domain present in the terminases. The terminase of l has been credited with helicase activity but the strand separation is not processive nor clearly dependent on ATP,43,88 so this activity does not imply that the terminase is a self-sufficient translocase. The most intriguing evidence of packaging-related binding comes from the T4 system: double-

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stranded specific binding could be localized to the ATPase domain,126 and there is evidence of a correlation between the state of the binding site and the state of the ATP-binding pocket. There are two possible functions of this DNA binding ability – engaging the DNA during translocation and/or facilitating cleavage by the adjacent nuclease domain – both of which are compatible with the data. The f29 ATPase, which does not have a nuclease domain, also binds DNA and induces supercoiling of DNA loops, but the ATP dependence of this process is not clear,80 so this is not a conclusive link to ATP-dependent translocation. Finally, the optical tweezers result that Q-motif mutants of l are unusually sensitive to load105 established that the Q-motif is involved in tightly coupling nucleotide changes to conformational changes that translocate the DNA – meaning that the Q-motif is an important part of the mechanical pathway of force generation. It is clear from these experiments that, upon changes in nucleotide state, the ATPase in turn undergoes conformational changes. Whether these conformational changes directly move the DNA, or indirectly actuate another component, remains unclear.

4.22.4.4.2

The connector/portal is intimately involved in translocation and regulates the ATPase

The other motor component that has been proposed to actively participate in packaging is the portal/connector. As early as 1978 – before the symmetry state of the connector was known – Hendrix, in a remarkable chain of inductive logic, proposed that the connector exhibits sixfold symmetry, that the connector engages the DNA, and that the symmetry mismatch between the connector and capsid could allow the connector to rotate while translocating DNA into the capsid.57 The first two of these proposals were substantiated, and the third idea, unproven, became a paradigm of viral packaging, spawning a number of more detailed models.49,58,60,63,127,128 Recent experiments on portal mutants have shown an intricate interaction between the portal and the ATPase, giving credence to the idea that the portal may constitute the motor. The connector is intimately involved in the packaging process: it encircles the DNA during packaging; it binds DNA; it modifies the hydrolysis rate of the ATPase; and it is responsible for headful sensing in packaging termination. The position and organization of the connector are clearly compatible with DNA engagement, and connectors of packaged T4, T7, and f29 particles were shown to bind about 40 bp of a linear DNA molecule in footprinting assays of mature phage, proving a DNA-binding capability, albeit in a different part of the life cycle.129 A possible structural basis for this interaction was revealed by crystal structures of the SPP1 portal, which showed that the most constricted part of the channel – 18 A˚ measured perpendicular to the DNA axis – is defined by the ‘tunnel loops,’ 16-residue interhelical regions whose positioning depends on the bending of an adjacent alpha helix63 (at the junction of red and black in Figure 4(b)). In order to explore the nature of the connector–DNA interaction, the effect of portal mutations on packaging and hydrolysis by SPP1 proheads was studied.56,130,131 Three intriguing mutations were examined in great detail: two in the clip domain responsible for ATPase binding, and one located in the tunnel loop predicted to contact the DNA. None of the mutations

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Viral DNA Packaging Motors

were predicted to change secondary structure, and indeed none had an effect on prohead formation or ATPase binding. In contrast, all three mutations significantly reduced both ATP hydrolysis by the ATPase and the efficiency of packaging. This apparently regulatory function was interpreted as ‘cross-talk’ between the portal and the ATPase: the portal may assume one of several states, and that state has an effect on the ATPase. Moreover, the location of the tunnel loops involved in this coupling suggests that the DNA is also a participant in the communication. Following up on these experiments was the discovery that flexibility in the portal is essential for packaging.132 Using the crystal structure as a guide, cysteines were introduced into the stem helices of the SPP1 portal to permit reversible crosslinking of neighboring subunits. In oxidizing conditions, the portals formed covalently linked high-molecular weight oligomers. Interestingly, proheads with crosslinked portals are incapable of fully packaging DNA in a stable manner; however, packaging could be restored simply by exposing the complexes to reducing conditions, proving that removal of the crosslinks is sufficient for normal activity. Abrogation of packaging was not simply due to blocking of the channel, as particles crosslinked after maturation exhibited normal ejection kinetics. Hence it was proposed that crosslinking inhibits conformational flexibility in the portal that is essential for packaging itself. Integrating the conclusions of the crosslinking and mutational studies, it is clear that connector conformation and dynamics can affect both packaging and ATP hydrolysis. However, there is no evidence to date of communication in the opposite direction – no evidence that the ATPase can regulate, stimulate, or actuate the connector. This kind of communication would be essential in any kind of ATP-fueled packaging by the connector.

4.22.4.4.3

Putative connector/portal rotation

Given the evident importance of the connector in the packaging process, connector-centric DNA translocation mechanisms have been proposed. The 12-fold symmetry of the connector, with each subunit capable of forming specific interactions with the DNA (for example, the tunnel loops of SPP163 or the lysines of f2949,133), does not match the symmetry of the DNA, which is about 10.5 bp per turn. In order to reconcile this mismatch, DNA must rotate relative to the connector as it passes through. If the connector is fixed on the capsid, the DNA must rotate, which may pose severe topological difficulties if the DNA is prevented from rotating freely either outside (due to external DNA-binding factors) or inside the capsid (due to molecular crowding). Hence, the connector itself may have to rotate relative to both DNA and capsid during packaging. In 1978, Hendrix observed that the symmetry mismatch between the 12-fold connector and locally fivefold capsid vertex could provide a physical basis for the connector to rotate on the capsid.57 Informed with new data, such as finding hydrophobic residues at the connector–capsid interface which suggest that the system is lubricated,49 many authors have elaborated on this model.49,58,60,63,127,128 In this general family of models, nucleotide hydrolysis in the ATPase would cause a chain of conformational rearrangements that propagate to the connector, hence driving the DNA into the capsid in a tightly-coupled manner and simultaneously

forcing the connector to rotate. The ATPase may push directly on the DNA and the DNA may exert torque on the connector as it passes through; or the ATPase may push tangentially on the connector, thus rotating it, and forcing the DNA through the connector as a rotating nut pulls a bolt. Indeed, any packaging mechanism in which the connector performs tightly coupled mechanical work without any kind of slippage on the DNA should involve rotation. This hypothesis was recently tested both in vitro and in singulo. Mutants of T4 with a bulky fusion to the portal protein were produced.134 The additional domain is known to bind specifically to the capsid and, thus, was expected to prevent rotation of the portal. These mutants assembled properly and displayed normal packaging behavior, suggesting that portal rotation is not required for packaging. The more direct test of the rotation hypothesis utilized single-molecule fluorescence polarization tracking of the connector of f29.14 A single fluorescent dye was attached to the connector dodecamer; if the connector were to rotate during packaging, the polarization of the fluorescent emission would rotate along with it. Although single complexes packaged normally in magnetic tweezers, simultaneous observation by total internal reflection microscopy revealed no polarization change above the noise. Because the strength of the signal would depend on the exact orientation of the dye relative to the total internal reflection surface, six different dye locations on the connector were interrogated in this way. Any unidirectional connector rotation between 0.6 and 36 degrees per base pair is incompatible with the data. This experiment ruled out all extant proposals for unidirectional, packaging-coupled connector rotation. Although the connector does not rotate, the aforementioned symmetry mismatch between the DNA and the packaging complex may still require rotation – that is, rotation of the DNA relative to the rest of the complex. Alternatively, perhaps the DNA is not rigidly coupled to the portal. A singlemolecule mechanical experiment supports this idea. Atomic force microscopy of free connectors135 found that the connector is elastic at a force of 100–150 pN – the same scale as the forces involved in packaging.10,96 Such conformational flexibility, which may be the same flexibility that is required for packaging132 may allow the connector’s DNA-binding domains to accommodate the mismatches, and hence avoid accumulating any angular misalignment.136,137 If the connector is not tightly coupled to the DNA, what is its role in packaging? At the very least, the connector is essential for proper assembly of both the capsid138,139 and the packaging motor. In f29, the connector N-terminus is known to bind to the pRNA48,64,140,141 and may simply serve as a temporary platform for pRNA binding. After packaging is complete, the rest of the packaging motor disassembles, leaving the connector responsible for preventing DNA ejection after departure of the ATPase and before docking of the tail. This may explain the portal mutant phenotypes in which the number of stably packaged particles is drastically reduced.130,132 Mutations homologous to the tunnel loop mutations of SPP1 were produced in f29 and single packaging events were monitored in optical tweezers. The characteristics of packaging such as velocity and force sensitivity were unchanged, suggesting that the mutations must disrupt the process at a later stage, such as in preventing post-packaging

Viral DNA Packaging Motors

DNA leakage (Rockney Atz, Shuhua Ma, K. Aathavan, Carlos Bustamante, Jiali Gao, Dwight L. Anderson and Shelley Grimes, in preparation). The connector may assume a similar duty during packaging itself: It could act as a one-way valve, allowing DNA to be packaged into the capsid but restraining its exit.14 In fact, a recent paper by Jing et al.142 demonstrated that DNA driven by an electric potential can pass in only one direction through connectors inserted into a lipid bilayer membrane – the direction corresponding to packaging into a prohead – and provided evidence that, in partially packaged particles, the connector alone can prevent DNA escape from the prohead. Such a one-way function would assist the packaging motor in maintaining processivity when packaging against high loads or high internal pressures as the capsid fills. Given the apparent universality of connector structure, the elusive details of connector function may well prove to be fundamental in nature.70 The popular model for connector-driven packaging has been experimentally disproven. Moreover, there is no direct evidence that the ATPase signals or actuates the connector, but rather that the connector can regulate the ATPase. Given that the phage ATPases are phylogenetically clustered with other motors that directly engage their substrate, these facts point toward a consensus that the phage ATPase does constitute the active packaging motor, hydrolyzing ATP and driving the DNA, while activated and intricately regulated by the remaining components in the packaging complex. Indeed, if the ATPase could translocate independently of the prohead, it could move beyond the initiation site before loading onto the prohead and hence impair packaging, or perhaps even move to another cleavage site and trigger wasteful truncation of the DNA; hence, a regulatory connector/portal may be an entirely necessary partner for a DNA-translocating ATPase. Confirming these hypotheses will be a major goal of research in the near future as part of the quest to understand the exact mechanism by which the DNA is translocated.

4.22.4.5

Structural Models of DNA Translocation

Earlier, we reviewed the dynamics of packaging and its implications for the mechanism for packaging by f29; these data can now be complemented by new structural information about the packaging ATPases. In this section, we will discuss the structure of the packaging ATPase of T4 phage and review the structure and mechanism of closely related motors. By combining time-resolved information from single-molecule studies of the f29 packaging motor with atomic-resolution snapshots of the related motors, we can start to assemble, frame by frame, a molecular movie of the packaging mechanism.

4.22.4.5.1

ATP binding

The ASCE family shares a common core secondary structure consisting of five parallel beta strands. The loops at the ends of the beta strands bear residues that are crucial for ATP binding and hydrolysis, such as the phosphate-binding Walker-A motif, the metal-binding Walker-B motif, a catalytic acidic group, a polar Sensor or C-motif or motif III, and an arginine finger (reviewed in Refs. 3, 102, 103, 114). Typically,

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nucleotide is bound at the interface between two subunits, in a groove formed by the arginine finger of one subunit and the other critical residues from the partner subunit. ATP hydrolysis begins with nucleotide and metal ion positioning, followed by nucleophilic attack of a water molecule on the gamma-phosphate, guided by the catalytic acid. The sensor or C-motif is thought to sense the presence of nucleotide and couple nucleotide binding to conformational changes elsewhere in the protein; likewise, the arginine finger may communicate a change in nucleotide state to the adjacent subunit. In addition to a host of mutational studies, the T4 terminase crystal structure – the only extant structure of a dsDNA viral packaging ATPase – confirms this general structure for the ATPbinding site, although the arginine finger comes from the same subunit.28,35

4.22.4.5.2

Conformational changes coupled to ATP hydrolysis

Local conformational changes associated with ATP hydrolysis must propagate through the protein to the DNA. Several different mechanisms for coupling hydrolysis to translocation have been proposed based on crystal structures of various nucleic acid translocases (reviewed in Refs. 3, 143, 114). The T4 terminase was crystallized with very close contacts between the ATPase domain and the DNA-binding nuclease domain whereas in cryo-EM, the domains appear to be spatially wellseparated, leading to a proposal that a change in nucleotide state is coupled to switching between ‘tensed’ and ‘relaxed’ states, a movement equivalent to two base pairs28 (Figure 9(a)). This hypothesis was supported by experiments showing that mutations to the interface between domains reduced packaging but not packaging-independent ATP hydrolysis, presumably because switching between the tensed and relaxed states was disrupted.28 Moreover, the fact that the T4 terminase appears to utilize an intramolecular arginine finger suggests that interdomain, rather than intermolecular, motions accompany nucleotide turnover. This ‘inchworm’ model for DNA translocation is very similar to one inferred from crystal structures of FtsK, a close relative of the f29 packaging motor, which suggested a hinge motion between the alpha and beta domains of the protein due to change in nucleotide state.122 However, the f29 packaging motor contains neither an FtsK-like alpha domain nor a T4-like nuclease domain, hence ruling out such an inchworm mechanism in this virus, and casting doubt on the functional relevance of the hinge motion in FtsK translocation. Moreover, a strict inchworm mechanism, making identical contacts after each step, could not explain the 2.5-bp steps that correspond to single ATP turnovers in f29, nor the sensitivity of the motor to every tenth phosphate. Although some other molecular motors have been proposed to act as domain-hinge inchworms, in particular, the socalled monomeric SF1 and SF2 helicases,143 there exist other structurally-motivated models. It was recently proposed that f29 stores elastic energy in the beta sheet of the ASCE core,136 based on analogies with F1-ATPase.144 As an alternative example, the conformational changes in the Rho helicase are transmitted through a network of salt bridges within one subunit and between adjacent subunits.119

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ATP binding

T*

ATPase

N

T

D

7Å

Nuclease

T** T

N

T

E

C C

D

E

T

T

T*

T*

7Å

(a)

Relaxed

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ADP

ATP

Pi

(b)

Figure 9 Proposed mechanisms of DNA translocation. (a) Upon nucleotide binding to the ATPase domain, the terminase large subunit of T4 is thought to undergo domain motions allowing the nuclease domain (brown) to pull DNA by 7 A˚, or 2 base pairs. After one translocation step, a new motor–DNA contact must be made by subdomain II (gray), or by another subunit in the oligomer. (b) The RNA helicase Rho forms a ring (colored balls) with nucleotides bound at the interfaces (not shown); the ring encircles an RNA polymer (gray balls). The six RNA binding loops shown here form a spiral staircase tracking the RNA helix, with the height of each lever and the extent of interaction with the RNA corresponding to the state of its ATP-binding region (T ¼ ATP, T ¼ ATP tightly bound in preparation for hydrolysis, D ¼ ADP, E ¼ empty). Although the phage ATPases discussed in this chapter are five-membered rings that translocate DNA, they may operate via a similar mechanism. Part (a) adapted from Williams, R. S.; Williams, G. J.; Tainer, J. A. A charged performance by gp17 in viral packaging. Cell 2008, 135 (7), 1169–1171, courtesy of J. Tainer. Part (b) inspired by Patel, S. S. Steps in the right direction. Nature 2009, 462 (7273), 581–583. Copyright by Nature.

Bioinformatics has implicated the C-motif in linking product release to conformational change.123 Some C-motif mutants of T4 are capable of binding and hydrolyzing ATP, but not releasing product, which can be explained if coordination between DNA binding and nucleotide state had been disrupted.123 Combining this idea with the single-molecule result that implicates the adenine-proximal Q-motif in force generation,105 it seems that the ATP binding pocket – a dense network of hydrogen and ionic bonds – is a tightly coupled system that reacts in specific ways in response to a change in nucleotide state. Conversely, the ATP binding site itself must catalyze hydrolysis or change affinity in response to DNA translocation. In some molecular motors, the ‘power stroke’ – the force-generating conformational change – coincides with ATP binding, largely due to the zippering of ionic bonds around the phosphate groups.101,145 However, in the f29 packaging motor, the power stroke is not ATP binding, but rather, most likely, post-hydrolysis phosphate release.11 Interestingly, the rotary motor F1-ATPase combines the two models: it performs two strokes per ATP turnover, corresponding to ATP binding and phosphate release.145,146 The terminases of l and T4 may follow either model, or perhaps stroke at another step in the chemical cycle.

4.22.4.5.3

DNA binding

The conformational changes in the protein must be efficiently passed on to the DNA through DNA-binding residues. The T4 terminase has been shown to bind DNA through the ATPase domain,126 but these contacts are not evident from the crystal structure.28 Although the terminase was crystallized in the absence of DNA, modeling revealed likely contacts between

the nuclease domain and the DNA via loops containing specific arginines and lysines.28 These contacts were distinct from the binding sites used in cleavage. Such an electrostatic mode of contact could explain the phosphate interaction observed in f29 and is also consistent with previously published observations of nucleic acid binding by single-stranded translocases such as phage f12 protein P4,147 bovine papillomavirus E1 helicase,120 and Rho helicase,119 although the structures and proposed mechanisms of those motors are quite different. The latter two proteins were crystallized as substrate-bound oligomeric rings with subunits in each of several different nucleotide states, so they may provide the most biologically relevant information. The DNA-binding residues are typically located in a flexible loop, making specific contacts with the backbone, and with the adjacent residues sometimes helping to stabilize the complex via side-chain and main-chain hydrogen bonds.103,148 The DNA binding loops of the subunits in the ring form a ‘spiral staircase’ that tracks the substrate backbone, such that several subunits simultaneously make contact with the substrate. The position of a binding loop is dependent on nucleotide state, and the spiral of binding loops is thought to move as a wave, pushing the DNA as hydrolysis propagates around the ring (Figure 9(b)). The specific relationship of nucleotide state and loop state gives rise to the polarity of translocation.119 Moreover, stepping by the motor is coordinated also because the affinity of the loop for DNA varies with nucleotide state. For the f29 ATPase, as well as for a number of other RecA-like hexameric nucleic acid translocases, the ATP-bound state has highest affinity for the polymer while the ADP-bound and apo states have much lower affinity.11,103

Viral DNA Packaging Motors 4.22.4.5.4

Intersubunit coordination

The wave mechanism used by the related ring ATPases requires a high degree of coordination between subunits to enable efficient translocation. This communication in the hexameric helicases, among other members of the ASCE division, is mediated by nucleotide bound at the interfaces between subunits.102,103,114 Therefore, the coordinated and sequential firing of the DNA binding loops in the hexameric helicases should be accompanied by a change in nucleotide state that proceeds, sequentially, around the ring. However, the T4 terminase structure provides no clear mechanism for intersubunit communication, because it lacks an intermolecular arginine finger.35 If there is coordination, as there must be if each subunit has only one DNA-binding site, the signal may be transmitted through the DNA itself. In contrast, the singlemolecule data on f29 contains strong signals of nucleotidemediated coordination: at least four subunits bind ATP before a physical step takes place, and four physical steps take place before the next round of ATP binding can begin.12 The extent of this coordination – whether hydrolysis, phosphate release, or ADP release are also synchronized and temporally separated – has yet to be fully explored.

4.22.4.5.5

Constructing a molecular model for the packaging motor of /29

We now outline a potential model for packaging that integrates data from the packaging motors with inferences from other ATPase molecular machines (Figures 7, 8, and 9) (for a more detailed model, see Refs. 66, 136). The packaging ATPase motor is a five-membered nearly-planar ring that encircles the DNA and binds ATP at the interfaces between the subunits. In this state, the DNA-binding loops or residues are positioned such that they have high affinity for the substrate, although each may not actually contact the DNA at this time. Once at least four ATP molecules are bound, one of the subunits hydrolyzes its ATP and subsequently releases inorganic phosphate while moving the DNA-binding motif over a distance of about 2.5 base pairs. This DNA-binding motif, utilizing any or all kinds of interaction – electrostatic attraction, electrostatic repulsion, hydrophobic interaction, hydrogen bonding, van der Waals bonding, etc. – pushes the DNA by about the same distance. At the end of the stroke, the change in nucleotide state has two effects: it causes the adjacent subunit to hydrolyze its ATP, release phosphate, and undergo a stroke, and it also reduces the affinity of the first subunit for DNA, hence allowing the DNA to move with the second subunit. The chain of hydrolysis proceeds around the ring until four steps, adding to 10 base pairs, have been taken. The four ADP molecules must then be exchanged for ATP while the motor does not stroke; exchange is probably sequential, with the energetic cost of releasing ADP from one binding pocket being balanced by the energetic benefit of binding ATP to another.12,136 During the extended stationary period (including ATP binding and at least four other steps), the fifth subunit, being in the highaffinity ATP-bound state, grips the DNA tightly through an electrostatic interaction with the phosphate backbone. Because this bond is ionic, the total distance packaged in a full cycle is rectified to be exactly 10 base pairs even though slight misalignments or slips take place during the burst. Once the four

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other subunits bind ATP, the hydrolysis cycle can begin again – triggered by the fifth subunit, which is making the phosphate contact that is known to regulate entry into the burst phase. The entire process is likely allosterically activated by the connector, which also acts as a one-way valve to prevent backward slipping. This model explains the data we have reviewed here for f29, but it raises some important biophysical questions. For example, what is the extent of coordination in the system – are hydrolysis, phosphate release, or ADP release also synchronized and temporally separated? What is the exact nature and geometry of the motor–DNA interaction? What is the role and physical origin of the unique fifth subunit? And more generally, how universal is this mechanism, among the packaging motors and among the DNA translocases as a whole?

4.22.5

What are the Organization and Physics of the Genome during and after Packaging?

The ultimate purpose of the packaging phenomena discussed thus far is to compact the viral genome inside the capsid. Compaction of DNA to the near-crystalline densities found in mature viral particles requires overcoming a significant energetic barrier, the size of which depends crucially on the physics of condensed DNA (reviewed in Refs. 149, 150). Moreover, it has been suggested that the high energy of packaged DNA assists the ejection of DNA from the particle during the subsequent infection process; thus, the stored energy can have crucial biological consequences.151 Here we discuss the dynamics of packaging into near-full capsids, and the organization and energetics of the packaged DNA.

4.22.5.1

Dynamics of Packaging at High Internal Filling

f29 packages at a maximum velocity of about 100 bp s–1, but it slows down as more DNA is encapsidated,10 similar to the decrease in packaging that arises if an external force is applied to the DNA being packaged (Figure 5(b)). This observation suggests that there is an internal force, resisting packaging, that builds in the capsid as the DNA is packaged. With some assumptions, Smith and coworkers were able to use the dependence of the packaging velocity on externally applied force to estimate this internal force from the measured packaging velocity.96 The total effective force – the sum of the external load and the inferred internal force – at which packaging velocity drops to zero is 110 pN. When the motor is unopposed by an external force, it reaches these slow packaging velocities only at the end of packaging its full 19.3 kbp genome, suggesting that B100 pN is the internal force resisting packaging when the entire f29 genome is packaged. It is experimentally difficult to measure the physics of DNA compressed to such high densities; thus, this indirect estimate of the mechanical energy needed to package the genome has provided a useful basis for improving theoretical models of the physics of DNA confinement.149 Similar arguments have been used to estimate the energetic cost of DNA confinement inside the capsid for T4 and l,87,92 and the energy scales and internal forces are all comparable. T4, l, and f29 have

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significantly different genome sizes, but their capsid volumes scale proportionally, suggesting that, perhaps, the physics of DNA confinement constrains the co-evolution of genome size and capsid volume.

4.22.5.2

Capsid Dynamics at High Internal Filling

Measurements of packaging velocity as a function of capsid filling have also revealed structural changes in the capsid during packaging. Volume increase concomitant with capsid expansion73 should reduce the free energy of the confined DNA and thus reduce the energetic cost of packaging more DNA, thereby lowering the resistance to packaging. In l, the velocity of packaging is a constant 600 bp s–1 until the genome is about 20% packaged (Figure 5(c)), at which point the velocity begins to drop.92 The drop is interrupted by a sudden increase in velocity at about 30% filling, which coincides with biochemical observations of the timing of capsid expansion.43 In simulations of packaging, the energy of packaged DNA is particularly sensitive to the volume and geometry of the capsid,152 suggesting that even a minor change in the capsid structure could have an effect on packaging velocity. A second structural change was also apparent in the single molecule experiments: at about 90% filling, many packaging complexes suddenly accelerated to the maximum velocity, which can be explained if the DNA were no longer strongly confined. Because the in singulo experiment lacked the capsid-stabilizing accessory protein gpD, the acceleration was interpreted as capsid rupture. Moreover, the ‘internal pressure’ at which the capsid ruptures is an indication of the capsid strength. In this way, optical tweezers can be used as a tool to probe both the dynamics and structural mechanics of the capsid.92,96

4.22.5.3

Energetics of DNA Confinement

The energy of DNA confined in the capsid is determined by contributions from electrostatic self-repulsion, bending strain, twist strain, volume exclusion, and entropy (reviewed in Ref. 149). The dominant factor is thought to be electrostatic, although entropy may also be significant.153 In order to experimentally assess the electrostatic component of the confinement energy, Fuller et al. examined packaging in a variety of different buffer conditions containing different species and concentrations of cations.154 After controlling for the chemical effects of the different cations on turnover of the packaging motor itself, the internal force could be calculated. This internal force generally decreased with cation valency and concentration, as would be expected due to electrostatic shielding, but this force was uniformly much higher than the quantitative predictions of existing models.155 This discrepancy points to an incomplete understanding of either the mechanism by which packaging is slowed, or the physics of DNA itself. The physics of the confined DNA can be inferred by studying another step in the viral life cycle: DNA ejection. In ejection, the internal forces that drive the DNA out of the capsid must work against some external forces such as osmotic pressure (reviewed in Refs. 150, 156). By tuning the osmotic

pressure difference between the interior of the capsid and the outside environment (by varying the concentration of polyethylene glycol), and monitoring the dynamics of ejection, those ‘internal pressures’ can be estimated.95,157–162 The inferred pressures are in the range of a few tens of atmospheres. It should be noted that the functional equivalence of polymer osmotic pressure and DNA internal forces has not been proven. In order to address this concern, isothermal titration calorimetry of ejection by l has been used to directly measure the heat of ejection.163 Interestingly, although the heat released is consistent with theoretical estimates, the contribution of entropy is, surprisingly, opposed to DNA ejection. The effect was attributed to a decrease in entropy of solvation as the DNA leaves the crowded capsid interior and acquires a water layer. This counterintuitive finding highlights the limitations of coarse-grained models of DNA compaction. More experimental and theoretical work is needed to fully understand the physical nature of the packaged viral particle.

4.22.5.4

Organization of Packaged DNA

The energy of the packaged DNA is also expected to depend on its arrangement inside the capsid. Minimization of bending energy suggests that the DNA, having a persistence length of 50 nm,164 would prefer to bend as gradually as possible inside the capsid, forming loops the size of the capsid inner diameter.165,166 At the same time, electrostatic energy is likely to be minimized by a hexagonal close-packed configuration.166,167 Images of fully assembled particles seem to confirm this intuition: When viewed along the connector/portal axis, a rotationally symmetric, radially periodic pattern in the DNA electron density can be seen (Figure 10),62,168–173 and these patterns were initially interpreted as spools. However, on closer inspection of some phages, this interpretation has been challenged. The periodic pattern in f29 is visible from all angles and, more significantly, the pattern does not emerge until greater than 90% of the genome has been packed.168 Hence, this local order need not reflect any long-range organization such as spooling (except for phages that have an axial core such as T7).168,173 Indeed, DNA inside P2 and P4 phages is capable of forming a wide variety of knots174,175 when the cohesive ends are allowed to anneal, suggesting a great deal of heterogeneity in DNA organization between particles. This heterogeneity suggests that the internal forces, and thus the packaging dynamics, among an ensemble of single particles may vary. In summary, while the packaging of DNA inside the particles may lead to some close packing of the local segments of the DNA, models in which the DNA is an orderly spool have not been experimentally validated.

4.22.6

How is Packaging Terminated?

The apparent heterogeneity in genome topology for a given virus type is not necessarily accompanied by variation in packaged genome length. Uniformity in packaged length from particle to particle has the dual effect of ensuring that every particle contains the full complement of genes while also minimizing wasteful energy consumption during assembly.

Viral DNA Packaging Motors 4.22.6.2

(a)

(b)

(c)

(d)

Figure 10 3-D cryo-EM reconstruction of mature f29 particles reveals DNA organization. A periodic pattern is apparent in the DNA density of cross-sections from the top (a) and side (b) of the reconstructed particle. 3-D views of the reconstruction (c) and (d) reveal individual layers of DNA, in addition to rings of DNA close to the connector (arrows). Reproduced from Comolli, L. R.; Spakowitz, A. J.; Siegerist, C. E.; Jardine, P. J.; Grimes, S.; Anderson, D. L.; Bustamante, C.; Downing, K. H. Three-dimensional architecture of the bacteriophage f29 packaged genome and elucidation of its packaging process. Virology 2008, 371(2), 267–277 and provided by L. Comolli, A. Spakowitz, and K. Downing.

Not surprisingly, terminase phages possess mechanisms to assure the packaging of a specific length of DNA. To this end, phages employ one of several strategies to terminate packaging at the appropriate point (reviewed in Refs. 4, 19). In this section, we will summarize these mechanisms, with particular focus on the role of the packaging motor in termination.

4.22.6.1

Termination of Packaging of Unit-Genome DNA

Phages that replicate DNA in single-genome segments, such as f29, reach a natural termination point when the entirety of the DNA molecule has been packaged (Figure 1(a)). Although packaging slows as the capsid fills, the velocity after one genome’s worth of DNA has been packaged is about 5% of the maximum velocity.10 When the end of the DNA has been reached, the motor (in the presence of nucleotide) continues to hold onto the DNA and the capsid until a tail complex displaces it.11 While the pRNA and ATPase are dissociating from the capsid, it is thought that the connector prevents the DNA from being ejected. After the tail assembles and binds, a short length of DNA slips out from the capsid into the tail,82 where the terminal protein is bound by the tail, yielding a stable, mature particle.

441

Sequence-specific Termination of Packaging of Concatemeric DNA

Viral species that replicate DNA as concatemers must resolve single genomes from the long DNA, a process that involves both halting translocation and performing endonucleolysis (Figure 1(b)). Phages that cut site-specifically, such as l, use a dimer of terminase small subunits to recognize a sequence or set of sequences (reviewed in Ref. 43). Interestingly, in l, several properly spaced sequences are required for termination; moreover, these sequences are not identical to the sequences used at packaging initiation. In terminal cleavage, cutting takes place at the so-called cosN site but is assisted by the upstream cosQ site, which is required for stopping translocation, and the I2 site, which stimulates cutting. Once the proper site has been recognized by the small subunit oligomer, the terminase large subunit nicks the DNA on each strand. The terminase holoenzyme then detaches from the capsid while remaining bound to the DNA, and is competent for packaging the attached DNA into a fresh prohead. Although cleavage minimally requires these sequences, the efficiency of the process depends on another factor: the amount of DNA packaged. When the l genome is shortened considerably, cleavage is reduced, suggesting that the cleavage is allosterically activated or stimulated by the presence of a filled head.176 In this way, the end of the packaging process also involves the interaction of several dynamic motor components.

4.22.6.3

Headful Termination of Packaging of Concatemeric DNA

Some phages – for example T4, P22, and SPP1 – package approximately, but not exactly, one genome length of DNA before cleavage. The exact length of DNA packaged varies from particle to particle, with the average packaging length dependent on the size of the capsid,177 suggesting that the packaging complex somehow recognizes that the capsid is full before cleaving. This phenomenon is called headful termination. If the headful mechanism is not activated, multiple DNA molecules can be packaged by T4.178 Mutational screens found that, in P22 and SPP1, the portal is responsible for headful sensing. Single-residue substitutions in the portal can increase or decrease packaging length179,180 and the effect of mutations seems to be additive. A structural basis for headful sensing has become apparent in recent years. Comparison of 18 A˚ structures of isolated wild-type and headful-defective portals of SPP1 showed significant differences in the crown domain.181 The recent cryo-EM reconstruction of the portal of mature P22 particles62,182 clearly shows that the portal makes contact with the encapsidated DNA, via those same crown domains. Moreover, comparison of this structure with the free portal reveals major structural differences in the crown region – differences that are attributed to the interaction with the highly pressurized DNA (Figure 4(c)). Thus the portal crown domain is likely to be the headful sensor; indeed, f29, which has no documented headful cleavage, has a significantly reduced crown domain compared to P22.183 Because the distal end of the portal is positioned to contact the terminase, it was proposed that conformational changes in the connector

442

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crown may propagate through the protein and ultimately to the terminase nuclease domain to actuate the cleavage. This hypothesis is corroborated by the fact that a specific mutation to the ATPase-binding clip domain – not the headful-sensing crown – also changes the length of packaged DNA.56 Because the nuclease domain of the terminase must be reoriented to switch from translocation to cleavage mode,28 packaging must be stopped before cutting can begin. This idea supports another important function of the connector/portal: as a headful-triggered regulator of translocation, which would explain the ‘cross-talk’ with the ATPase discussed earlier in this chapter.56 Moreover, the central role of the portal in headful cleavage suggests that it may also be involved in assisting sequence-specific termination for phages such as l. It is likely that dissecting the termination process will reveal much about the connector–ATPase interaction.

developed high resolution optical tweezers – techniques already used in the study of packaging dynamics – should provide an unprecedented view into the angstrom-scale dynamics of the packaging motors. Moreover, the combination of these experimental techniques on specific motor mutants105,106 or specific modifications of the nucleic acid substrate66 promises to provide the structural origin of a variety of specific dynamics. Of particular interest should be the exact structural components (i) that engage the DNA, providing stability and processivity and transducing force, (ii) that couple conformational changes in the well-studied ATP binding pocket to the domains that actually translocate the DNA, and finally (iii) that give rise to communication and coordination between subunits and create the small kinetic differences between the identical subunits.

4.22.7.2

4.22.7

Conclusions and Future Directions

Viral genome packaging has been an exciting avenue of biophysical research for many decades, and these studies have both yielded new insights into molecular motor dynamics and spawned a wide variety of new biophysical techniques. The sheer volume of work in this field prohibits covering all of the excellent work done and we have endeavored, in this chapter, to cover only the core mechanisms and phenomena associated with the basic core stages in viral genome packaging in the tailed bacteriophages: packaging initiation, DNA translocation, and termination. Tremendous advances have taken place in all of these areas, and clear pictures are starting to emerge for the functional role of the different components of the packaging complex. With molecular pictures starting to take shape, an entirely new class of more subtle and fundamental questions can now be asked. And, while any prediction of the future of research is likely to be far from the mark, we conclude our review by presenting some speculative thoughts on what these new questions may be.

4.22.7.1

How Do Structural Features Dictate and Shape Structural Dynamics?

High resolution optical tweezers studies, modified DNA studies, and high resolution structural snapshots have provided a unique picture of the mechanism of DNA translocation in a variety of bacteriophages. However, the exact structural features responsible for these dynamics have not been finalized. For example, specific biophysical functions – subunit coordination, catalysis rates, or packaging velocities – have not yet been conclusively and uniquely linked to individual sequence motifs – loops, beta sheets, or specific residues in the motor. Of course, this challenge is not unique to the packaging motors; rather, a detailed understanding of how specific structural motifs give rise to functional dynamics is really only starting to emerge in a few key molecular motors, such as RNA polymerase, DNA polymerase, kinesin, and myosin. However, the combination of high resolution static snapshots from crystallographic structures in combination with the one-dimensional dynamic measurements by the recently

How General are the Structural Dynamics in the Packaging Motors?

As detailed pictures of the structural dynamics emerge in a variety of packaging motors, it will be possible to address another interesting question: how general is the packaging mechanism? While there are many differences among the bacteriophage packaging machineries, bioinformatic studies point toward the common origin of these components and their underlying similarity. However, it is not yet clear that this core structural similarity leads to similar packaging dynamics. Despite the fact that the large terminase subunits and the ATPases of the f29-like bacteriophages share the basic structural features common to many ATPases and the fact that their final biological functions are essentially identical, it need not be the case that the fundamental dynamics of these two groups of packaging machines are the same. Moreover, it is possible that such differences may not occur at the single subunit level; the way in which the subunits are coordinated could easily be different between different bacteriophages. Low resolution optical tweezers studies of l and T4 have already revealed differences in basic dynamic parameters such as packaging velocity and force dependence. Extending these studies to higher resolution should immediately reveal whether or not the highly coordinated burst-dwell mechanism of f29 is common to these phages. In the longer term, the type of mutation-based studies discussed above will provide a very useful data set for comparative studies, allowing the field to address questions such as (i) what amino acids determine differences in packaging velocities (i.e., what are the molecular throttles); (ii) what structural features determine the difference in force dependence; and finally (iii) is subunit coordination a necessary component of packaging with a ring motor?

4.22.7.3

Concluding Thoughts

The essential functions of the cell involve a wide variety of remarkable mechanical processes, and of these phenomena, genome compaction by tailed bacteriophages is arguably one of the most amazing. Over the past decades, researchers have gone from simple visualization of these remarkable systems to full dissection of the molecular mechanisms of packaging. The molecular pictures that will emerge from the studies to come

Viral DNA Packaging Motors

promise not only to complete our molecular understanding of the genome compaction process but also, more generally, to provide insights into the basic design principles that govern the evolution and dynamics of molecular motors.

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