The Structure of the ATPase that Powers DNA Packaging into Bacteriophage T4 Procapsids

Molecular Cell

Short Article The Structure of the ATPase that Powers DNA Packaging into Bacteriophage T4 Procapsids Siyang Sun,1 Kiran Kondabagil,2 Petra M. Gentz,1,3 Michael G. Rossmann,1,* and Venigalla B. Rao2,* 1

Department of Biological Sciences, Purdue University, 915 W. State Street, West Lafayette, IN 47907, USA Department of Biology, The Catholic University of America, 620 Michigan Avenue NE, Washington, DC 20064, USA 3 Present address: Department of Biochemistry, Microbiology, and Biotechnology, Rhodes University, Grahamstown, Eastern Cape 6140, South Africa. *Correspondence: [email protected] (M.G.R.), [email protected] (V.B.R.) DOI 10.1016/j.molcel.2007.02.013 2

SUMMARY

Packaging the viral genome into empty procapsids, an essential event in the life cycle of tailed bacteriophages and some eukaryotic viruses, is a process that shares features with chromosome assembly. Most viral procapsids possess a special vertex containing a dodecameric portal protein that is used for entry and exit of the viral genome. The portal and an ATPase are parts of the genome-packaging machine. The ATPase is required to provide energy for translocation and compaction of the negative charges on the genomic DNA. Here we report the atomic structure of the ATPase component in a phage DNA-packaging machine. The bacteriophage T4 ATPase has the greatest similarity to monomeric helicases, suggesting that the genome is translocated by an inchworm mechanism. The similarity of the packaging machines in the double-stranded DNA (dsDNA) bacteriophage T4 and dsRNA bacteriophage f12 is consistent with the evolution of many virions from a common ancestor. INTRODUCTION Most bacteriophages (Black, 1988) and some eukaryotic viruses (Newcomb et al., 2005) assemble empty procapsids into which their genomes are subsequently packaged. The packaging machine generally consists of a dodecameric portal protein (head-tail connector) and an ATPase at one of the five-fold vertices of the procapsid. There are a number of stages in the process of packaging a viral genome into a procapsid (Figure 1A). First, the beginning of the DNA genome has to be threaded into the packaging machine. Then, the energy of ATP hydrolysis must be converted to mechanical energy, which drives the genome into the empty or partially filled procapsids. Upon completion of packaging, the ATPase component dissociates and links the DNA still associated with it to another empty procapsid. Finally, the termination of packag-

ing has to be signaled when the head is full (Chang et al., 2006; Lander et al., 2006) and a seal is created to keep the genome within the head. The DNA of most tailed phages is concatenated while replicating in the host cell. For these viruses, a nuclease component of the packaging machine cleaves the DNA when the head is full. For instance, on average, about 1.02 genomes are packaged into each bacteriophage T4 capsid with ends that are different for each mature particle (Streisinger et al., 1967). The only published atomic structure of a dsDNA phagepackaging motor component is that of the f29 portal assembly, which consists of a wider end (69 A˚) that is inserted into the head and a narrower end (33 A˚) that protrudes out of the capsid (Simpson et al., 2000). It is 75 A˚ long and has a central 36 A˚ diameter channel, lined with a helices with exposed negative charges, allowing the smooth passage of dsDNA. Cryo-electron microscopy studies have shown that the portal of the tailed phages T4 (Leiman et al., 2004), SPP1 (Orlova et al., 2003), P22 (Chang et al., 2006; Lander et al., 2006), and 315 (Jiang et al., 2006) have structures similar to the f29 portal, suggesting a common evolutionary origin for the packaging motor. Models for the mechanism of viral dsDNA translocation, which assume directional rotation of the portal powered by ATP hydrolysis, have been proposed based on the symmetry mismatch between the five-fold capsid, the dodecameric portal, and the ten-fold screw symmetry of DNA (Hendrix, 1998; Simpson et al., 2000). While the structural features of the portal are consistent with this model, portal rotation has not been demonstrated. Recent results showing that DNA packaging remains functional when the T4 portal is attached to the capsid argue against a rotating portal (Baumann et al., 2006). Another well-studied example of phage-packaging motor is that of the dsRNA bacteriophage f12, which utilizes a hexameric ATPase that functions both as a portal and as a single-strand RNA (ssRNA) translocase (Mancini et al., 2004). Structural snapshots of various conformations of the motor suggest that an RNA binding loop pushes the ssRNA through the hexameric ring of ATPase subunits that hydrolyze ATP by a cooperative and sequential mechanism. The prolate icosahedral bacteriophage T4 procapsids, about 1200 A˚ long and 860 A˚ wide (Fokine et al., 2004),

Molecular Cell 25, 943–949, March 23, 2007 ª2007 Elsevier Inc. 943

Molecular Cell The DNA-Packaging Machine of Bacteriophage T4

Figure 1. The T4 DNA-Packaging Machine (A) A schematic diagram shows the T4 procapsid while being filled with DNA. The portal protein, gp20, forms a dodecameric head-tail connector through which the genome enters the procapsid and exits the mature phage. The stoichiometry of the large terminase gp17 oligomer is suggested to be pentameric or decameric. The amino-terminal domain of gp17 has ATPase activity, whereas the carboxy-terminal domain has nuclease activity. There are probably eight to ten gp16 monomers in the small terminase oligomer. The small terminase functions to enhance the ATPase activity of the large terminase. (B) A ribbon diagram shows the gp17 N360-ED mutant structure. Subdomain I consists of the nucleotide-binding motif (Rossmann et al., 1974). The strands in the b sheet (colored, in sequence, red, orange, yellow, green, cyan, and blue) follow a 324516 topology, as in most other translocating ATPases. The ATP molecule is colored purple. The loop associated with adenine binding (purple) changes its conformation in the apo structure (yellow). The conserved ATP-binding subdomain I is spatially distinct. The N and C termini of gp17 N360-ED that form subdomain II may be a part of the Cterminal nuclease domain of gp17. The figure was generated with the PyMOL program (DeLano, 2002).

package a 171 kb genome. The DNA-packaging machine associated with the procapsids consists of the dodecameric portal (gene product [gp] 20, 61 kDa), the large terminase complex (gp17, 70 kDa) and the small terminase complex (gp16, 18 kDa) (Powell et al., 1990; Rao and Black, 1988). The stoichiometry of the large and small terminase complexes has not been established. The ATPase activity, required for DNA packaging, is resident in the amino-terminal domain (residues 1–360) of gp17, whereas the nuclease activity, required for the termination of DNA packaging, resides in the carboxy-terminal domain (residues 361–610) (Kanamaru et al., 2004; Mitchell et al., 2002). The small terminase complex on its own has neither ATPase nor nuclease activity, but it enhances 50-fold the ATPase and packaging functions associated with the large terminase complex (Leffers and Rao, 2000). Although there is strong biochemical evidence suggesting that the N-terminal gp17-ATPase powers the DNApackaging machine, it is unclear whether the ATPase merely provides free energy for packaging or actively drives DNA translocation through a relatively passive portal channel. Here we describe the atomic structure of a phage DNA-packaging ATPase to 1.8 A˚ resolution. Comparison of the structure with other ATPase motors suggests that the gp17-ATPase is analogous to monomeric helicases and likely provides the driving force for DNA translocation.

RESULTS AND DISCUSSION The gp17 ATPase Domain Structure Attempts to crystallize the wild-type, full-length gp17 or its two separate domains were unsuccessful. Several mutants of the N-terminal ATPase domain were generated that bound, but were unable to hydrolyze, ATP (Mitchell and Rao, 2006). Of these, the double-mutant N360-ED (D255E/E256D) could be crystallized as the apo protein and cocrystallized variously with ATP, ADP, and Mg2+, as well as combinations of these. The structure of the ATP complex was determined to 1.8 A˚ resolution (Table 1) and the other structures were determined by molecular replacement. The structure is a rather flat molecule measuring about 62 3 45 3 28 A˚. The primary folding motif has the topology of a nucleotide-binding fold, arguably the most ancient protein fold (Chothia et al., 2003; Rossmann et al., 1974), consisting of six adjacent, parallel b strands, usually associated with ATPase translocation proteins (Figure 1B). There are two spatially separated subdomains, with subdomain I (residues 105–313) being the nucleotide-binding fold, presumably responsible for the ATPase activity. Subdomain II consists of residues from both the N-terminal (residues 1–58) and C-terminal (residues 314–360) regions of the expressed polypeptide and, presumably, would be part of the nuclease component of gp17.

944 Molecular Cell 25, 943–949, March 23, 2007 ª2007 Elsevier Inc.

Molecular Cell The DNA-Packaging Machine of Bacteriophage T4

Table 1. Data Collection and Refinement Statistics ATP

ATP SeMet

ATP

ADP

ADP+Mg2+

Apo

home

14-BM-D

23-ID-B

23-ID-B

23-ID-B

23-ID-B

Data Collection X-ray source Wavelength (A˚)

1.5418

0.97910

1.20373

1.20373

1.03320

1.20373

Resolution (A˚)

2.0

3.3

1.85

1.8

1.8

2.3

Space group

P212121

P212121

P212121

P212121

P212121

P21

Unit cell (A˚)

a = 61.2, b = 62.4, c = 126.4

a = 61.0, b = 62.5, c = 126.3

a = 61.1, b = 62.3, c = 126.6

a = 61.1, b = 62.1, c = 126.6

a = 61.2, b = 62.4, c = 126.6

a = 38.7, b = 118.5, c = 47.5, b = 92.5

Unique reflections

29,395

14,144

41,159

45,358

45,523

18,177

Average redundancy

3.7

3.8

3.1

8.2

6.2

2.8

a

I/s

34.3 (3.2)

13.4 (5.6)

25.1 (2.7)

41.5 (4.3)

35.4 (6.2)

19.7 (1.7)

Completeness (%)

87.8 (63.8)

99.9 (100.0)

97.6 (97.7)

99.2 (97.3)

98.8 (99.4)

95.0 (88.9)

Rmerge (%)b

4.3 (37.1)

14.5 (32.0)

5.7 (42.5)

6.0 (43.9)

6.2 (37.2)

7.5 (52.3)

1.88

1.8

1.81

2.5

Rworking (%)

23.6 (31.3)

22.9 (31.8)

23.4 (28.6)

22.8 (34.4)

Rfree (%)e

24.8 (32.9)

23.7 (33.1)

24.1 (32.0)

27.8 (39.0)

Average B factor (A˚2)

44.0

42.5

42.2

74.2

Rmsd bonds (A˚)

0.005

0.007

0.009

0.008

Rmsd angels ( )

1.2

1.3

1.5

1.6

c

0.907 (iso) 1.021(ano)

Phasing power Refinement Resolution (A˚) d

a

Values in parentheses throughout the table correspond to the last shell. Rmerge = Sj I  < I > j / S I, where I is measured intensity for reflections with indices hkl. c Phasing power = [ j Fh(calc) j / phase-integrated lack of closure ]. d Rworking = SjjFobsj  jFcalcjj / SjFobsj. e Rfree has the same formula as Rworking except that calculation was made with the structure factors from the test set. b

stearothermophilus (Subramanya et al., 1996). Superposition of the various nucleotide-binding domains results in the approximate coincidence of the bound mononucleotide near the carboxy ends of the b strands in the b sheet (Figure S1 in the Supplemental Data available with this

Despite no detectable sequence identity, subdomain I superimposes well (Table 2) onto a variety of ATPases with different cellular translocation functions, such as bovine heart mitochondria F1-ATPase a- and b subunits (Abrahams et al., 1994) and PcrA helicase from Bacillus

Table 2. Structural Superpositions of gp17 N360-ED with Other ATPases Protein

Function

PDB Accession Number

Number of Equivalent Ca Atoms

f12 P4

dsRNA phage packaging motor

1W44

75

3.3

T7 gp4

hexameric helicase

1E0J

120

3.4

F1-ATPase a

rotary proton pump

1H8H

86

2.6

F1-ATPase b

rotary proton pump

1H8H

113

2.9

Rec A

recombination

1UBE

110

2.6

FtsH

hexameric metalloprotease

1IY0

111

3.3

PcrA+DNA

monomeric helicase

2PJR

138

2.7

Rec G

monomeric helicase

1GM5

133

2.8

Rmsd (A˚)

Molecular Cell 25, 943–949, March 23, 2007 ª2007 Elsevier Inc. 945

Molecular Cell The DNA-Packaging Machine of Bacteriophage T4

article online). The structurally based alignments (Figure S2) are mostly consistent with previous alignments based on amino acid sequence motif recognition (Mitchell et al., 2002). The ATPase Active Center The only completely conserved sequences among the aligned ATPases are the Walker A and Walker B motifs (Walker et al., 1982) (Figure S2). Their identification by structural alignment is consistent with mutational analysis, demonstrating that they are essential for catalysis (Goetzinger and Rao, 2003). The catalytic center of these ATPases is created by the conserved Walker A (Gly-LysThr) and Walker B (Asp) motifs and a catalytic glutamic acid residue (Goetzinger and Rao, 2003) (Figures 2C and 2D). The function of these residues is to anchor the b and g phosphates of ATP relative to the Mg2+ ion in order to facilitate a nucleophilic attack on the ATP (Abrahams et al., 1994). The associated release of energy is translated into motion caused by the separation of the ADP and Pi binding sites (Figure 2D). The structure presented here is, however, one in which the Walker B Asp255 was mutated to a Glu, and the neighboring catalytic Glu256 was mutated to an Asp. In the gp17 N360-ED mutant structure, the Walker A Lys166 has altered its position from interacting with the b and g phosphates (as in active ATPase structures) to form a salt bridge with the mutant Glu255, causing an 2 A˚ shift of the main chain. In addition, the bound ATP in the N360ED mutant has altered its position by about 3 A˚ away from the active center (Figure S1). These conformational changes prevent Glu255 from binding a Mg2+ through a water molecule and Asp256 from binding the g phosphate through a water molecule, explaining why the N360-ED mutant is unable to hydrolyze ATP (Figure 2B). Consistent with the structural changes imposed by the mutant protein, no Mg2+ or water molecules were detected in either the ATP or ADP complex, although Mg2+ was included in the crystallization conditions. The structure of N360-ED, when complexed with ADP or ATP, is essentially the same with a root-mean-square deviation of only 0.16 A˚ between equivalent Ca atoms, indicating that the structure is stabilized by the ED mutations. However, the apo N360-ED structure is significantly different in the Cys125-Arg140 loop, which interacts with the adenine ring (Figure 1B). The movement of this loop is the result of ATP hydrolysis and, therefore, may be pivotal in translating chemical energy into mechanical energy. Comparison with Monomeric Helicases The gp17 ATPase domain shows greatest structural similarity to monomeric helicases (Table 2 and Figure 2E). In addition, whereas the catalytically important Glu immediately follows the Walker B motif in the gp17 ATPase domain and in monomeric helicases (Gorbalenya and Koonin, 1993), in many other ATPases it is derived from a sequence upstream of the Walker B motif (Abrahams

et al., 1994). As a consequence, in the gp17 ATPase and monomeric helicases, there is a trans-peptide adjacent to the Asp in the Walker B motif, whereas there is a cispeptide in this position when the catalytically important Glu is upstream of the Walker B motif in order to place the upstream Glu close to the active center. A conserved feature between the gp17 ATPase domain and monomeric helicases is the TTT sequence (285–287) ‘‘motif III,’’ which is important in monomeric helicases for sensing the state of ATP in the catalytic pocket (Mitchell et al., 2002; Soultanas et al., 1999). Another feature of the gp17 ATPase domain and monomeric helicases is that the ‘‘switch’’ residue Arg162 in gp17 that interacts with the g phosphate is in the same polypeptide as the other catalytically important residues (Mitchell et al., 2002; Soultanas et al., 1999), whereas in oligomeric ATPases, the spatially equivalent residue is provided by a neighboring subunit (Abrahams et al., 1994). Helicase translocation is probably driven by an ‘‘inchworm’’-type mechanism requiring two distinct DNAbinding sites, which alternatively attach and detach from the substrate while undergoing a spatial translocation (Velankar et al., 1999). In the case of monomeric helicases, the two binding sites are each associated with a nucleotide-binding fold, and the translocation is achieved by a hinge motion between the two domains upon ATP hydrolysis (Singleton et al., 2001; Velankar et al., 1999). Because of the structural similarities between gp17 ATPase and monomeric helicases, translocation of the dsDNA into T4 procapsids might also be achieved by an inchworm mechanism. The two distinct DNA-binding sites required for each translocation cycle could come from either the same subunit as in monomeric helicases or from neighboring subunits as in the packaging motor of f12. Recent results (T.I. Alam and V.B.R., unpublished data) suggest that the N360 domain of gp17 can bind dsDNA, but the C-terminal nuclease domain of gp17 is unable to form a stable complex with DNA. This would imply that the C-terminal domain of gp17 primarily presents the catalytic residues required for cleavage of DNA in order to initiate or terminate packaging (Rentas and Rao, 2003), leaving the DNA-binding function to the N-terminal domain. However, as these results demonstrate that there might be only one DNA-binding site on each gp17 molecule, it is possible that the inchworm mechanism operates between two neighboring gp17 subunits associated with the site of the current ATP hydrolysis. A Mechanism for DNA Packaging Although gp17 presumably forms a ring surrounding the DNA in the T4 procapsid structure, the stoichiometry is unknown. The structure of B-form DNA contains ten base pairs per complete turn of the helix. Hence, if it were assumed that there were five gp17 monomers per ring, and since the hydrolysis of one ATP molecule transports approximately two base pairs into the procapsids, each gp17 monomer would detect the same DNA structural component as the neighboring gp17 after translocation

946 Molecular Cell 25, 943–949, March 23, 2007 ª2007 Elsevier Inc.

Molecular Cell The DNA-Packaging Machine of Bacteriophage T4

Figure 2. The Active Center of the gp17 ATPase (A) Stereodiagram of the ATP-binding pocket in the gp17 N360-ED mutant showing residues that interact with ATP (the figure was generated with the PyMOL program [DeLano, 2002]). Schematic drawing of the ATP-binding pocket in gp17 N360 (B) observed in the ED mutant, (C) before hydrolysis, and (D) after hydrolysis. The ATP, ADP, and Pi molecules are colored red, the catalytic residues are purple, the Mg2+ and water molecules are green, and the residues that bind the AMP fragment of ATP are black. K166 and T167 are part of the Walker A phosphate-binding ‘‘P’’ loop, and D255 belongs to the Walker B motif. Based on studies on homologous ATPases (Abrahams et al., 1994; Soultanas et al., 1999), E256, the catalytic Glu, activates a water molecule for nucleophilic attack on the pyrophosphate bond as shown in (C) (Subramanya et al., 1996). The g phosphate is then stabilized by R162, the ‘‘switch residue,’’ in a pentacoordinated transition state to facilitate the leaving of Pi after hydrolysis. In the ED mutant, K166 interacts with the mutated Walker B E255 instead of the b and g phosphates, and therefore Mg2+ and the two waters are not seen in the ED mutant structure. (E) The best Ca backbone superposition of gp17 N360-ED onto the monomeric helicase PcrA is shown in a stereodiagram. Note the superposition of the catalytic residues of N360 (red) and PcrA (blue).

Molecular Cell 25, 943–949, March 23, 2007 ª2007 Elsevier Inc. 947

Molecular Cell The DNA-Packaging Machine of Bacteriophage T4

of the DNA by two base pairs. This situation is the case for DNA packaging in f29, where there are five ATPase molecules situated in a ring surrounding the DNA (Morais et al., 2001; M.C. Morais, V.D. Bowman, D.L. Anderson, and M.G.R., unpublished data). In contrast, if the dodecameric portal were the basis of an inchworm translocation mechanism, as was suggested by Simpson et al. (2000), the portal would have to rotate by 12 to correctly align with the DNA structure for every ATP hydrolysis event. As portal rotation does not appear to be the case in T4 (Baumann et al., 2006), the translocation must be produced by a pentameric distribution of gp17 molecules around the base of the procapsids. Thus, the function of the portal appears to be merely passive, providing a negatively charged channel (Simpson et al., 2000) conducive for the passage of DNA without hindrance, whereas the capsid protein would not be able to provide this specialized function. The basic mechanism described here for dsDNA packaging is similar to that described for ssRNA packaging into bacteriophage f12 procapsids (Mancini et al., 2004), except that in f12 there is no dodecameric portal protein and there are six ATPases in a ring around the icosahedral vertices instead of presumably five ATPases at the special vertex of bacteriophage T4. A similar mechanism has also been proposed for bacteriophage f29 (M.C. Morais, V.D. Bowman, D.L. Anderson, and M.G.R., unpublished data). In this case there is a dodecameric portal protein of known structure, and the presence of five ATPases around the unique vertex has been well established (Morais et al., 2001; M.C. Morais, V.D. Bowman, D.L. Anderson, and M.G.R., unpublished data; Simpson et al., 2000). Considering the divergent evolution from a common ancestor for many phages based on their structural and functional similarities (Fokine et al., 2005; Jiang et al., 2006; Morais et al., 2004), the determination, by different procedures, of a similar genome packaging mechanism is a confirmation of each independent investigation. EXPERIMENTAL PROCEDURES Protein Preparation and Crystallization The gp17 N360-ED mutant was cloned and purified as described previously (Mitchell and Rao, 2006). The SeMet derivative was generated by methionine pathway inhibition (Doublie, 1997) and was purified in a similar manner as the native ED mutant. The ED mutant was concentrated to 5 mg/ml in 20 mM Tris-Cl (pH 8.5), 100 mM NaCl, 1 mM DTT, and 5% glycerol. One millimolar ATP or ADP was added to the purified N360-ED mutant. Crystals were grown by vapor diffusion in hanging drops at 20 C from 22% PEG3350, 0.2 M KNO3, and 8% glycerol. To obtain Mg2+-bound crystals, 5–25 mM MgSO4 was added to the crystallization mixture. Apo ED crystals were grown from the same conditions, but the crystals had a different morphology. Structure Determinations Data collection and refinement statistics are summarized in Table 1. Crystals were flash-frozen, and data were collected at 100K on a Rigaku rotating-anode ‘‘home’’ X-ray source and the Advanced Photon Source (APS) GM/CA-CAT 23-ID-B beamline. The APS BioCARS 14BM-D beamline was used to collect X-ray diffraction data near the

Se absorption edge. Data were processed with the HKL2000 program (Otwinowski and Minor, 1997). The space group of the gp17 N360-ED/ ATP crystals was consistent with P212121, with a = 61.2, b = 62.4, and c = 126.4 A˚, based on the initial cell parameters, subsequent scaling procedure, systematic absences, and eventual data determination. The Matthews coefficient of 2.8 A˚3/Da indicated that there was one molecule per crystallographic asymmetric unit. Structure factor phases were determined to 4 A˚ resolution using native home-source data from the ATP-bound complex together with a single wavelength diffraction data set of the SeMet ATP complex taken at the maximum f’’ Se absorption edge. Single-isomorphous replacement with anomalous scattering (SIRAS) was used for the initial phase determination. The phases were refined and extended to 2 A˚ using solvent-density flattening with the programs SHARP (de La Fortelle and Bricogne, 1997) and dm (Cowtan, 1994). The structure was built manually using the program O (Jones et al., 1991) and refined with the program CNS (Bru¨nger et al., 1998) to 1.88 A˚ resolution. The final Rworking and Rfree (using 5% of the data) were 23.6% and 24.8%, respectively. The isomorphous crystal structures of the gp17 N-domain complexed with ADP and with ADP+Mg2+ were refined to 1.8 A˚ and 1.81 A˚ resolution, respectively. Crystals of the apo N360 (space group P21, with a = 38.7, b = 118.5, c = 47.5 A˚, and b = 92.5 ) were not isomorphous with the ATP-complexed crystals. This apo structure was solved by molecular replacement using the ATP complex as a search model and the program CNS (Bru¨nger et al., 1998). The structure was also refined with the CNS program to 2.5 A˚ resolution. Supplemental Data Supplemental Data include two figures and can be found with this article online at http://www.molecule.org/cgi/content/full/25/6/943/ DC1/. ACKNOWLEDGMENTS We thank Ye Xiang for helpful crystallographic advice and Sheryl Kelly, Cheryl Towell, and Sharon Wilder for help in the preparation of the manuscript. We also thank the staff of the APS, BioCARS, and GM/ CA sectors for their help in X-ray diffraction data collection. Those facilities are supported by the U.S. Department of Energy and/or the National Institutes of Health. This work was supported by National Science Foundation grants to M.G.R. (MCB-443899) and V.B.R. (MCB-423528). Received: December 6, 2006 Revised: January 30, 2007 Accepted: February 12, 2007 Published: March 22, 2007 REFERENCES Abrahams, J.P., Leslie, A.G.W., Lutter, R., and Walker, J.E. (1994). Structure at 2.8 A˚ resolution of F1-ATPase from bovine heart mitochondria. Nature 370, 621–628. Baumann, R.G., Mullaney, J., and Black, L.W. (2006). Portal fusion protein constraints on function in DNA packaging of bacteriophage T4. Mol. Microbiol. 61, 16–32. Black, L.W. (1988). DNA packaging in dsDNA bacteriophage. In The Bacteriophages, R. Calender, ed. (New York: Plenum Press), pp. 321–363. Bru¨nger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., et al. (1998). Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921. Chang, J., Weigele, P., King, J., Chiu, W., and Jiang, W. (2006). CryoEM asymmetric reconstruction of bacteriophage P22 reveals

948 Molecular Cell 25, 943–949, March 23, 2007 ª2007 Elsevier Inc.

Molecular Cell The DNA-Packaging Machine of Bacteriophage T4

organization of its DNA packaging and infecting machinery. Structure 14, 1073–1082. Chothia, C., Gough, J., Vogel, C., and Teichmann, S.A. (2003). Evolution of the protein repertoire. Science 300, 1701–1703. Cowtan, K.D. (1994). ‘dm’: an automated procedure for phase improvement by density modification. Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography 31, 34–38. de La Fortelle, E., and Bricogne, G. (1997). Maximum-likelihood heavyatom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. Methods Enzymol. 276, 472–494. DeLano, W.L. (2002). The PyMOL molecular graphics system (http:// www.pymol.org). Doublie, S. (1997). Preparation of selenomethionyl proteins for phase determination. Methods Enzymol. 276, 523–530. Fokine, A., Chipman, P.R., Leiman, P.G., Mesyanzhinov, V.V., Rao, V.B., and Rossmann, M.G. (2004). Molecular architecture of the prolate head of bacteriophage T4. Proc. Natl. Acad. Sci. USA 101, 6003–6008. Fokine, A., Leiman, P.G., Shneider, M.M., Ahvazi, B., Boeshans, K.M., Steven, A.C., Black, L.W., Mesyanzhinov, V.V., and Rossmann, M.G. (2005). Structural and functional similarities between the capsid proteins of bacteriophages T4 and HK97 point to a common ancestry. Proc. Natl. Acad. Sci. USA 102, 7163–7168. Goetzinger, K.R., and Rao, V.B. (2003). Defining the ATPase center of bacteriophage T4 DNA packaging machine: Requirement for a catalytic glutamate residue in the large terminase protein gp17. J. Mol. Biol. 331, 139–154.

Mitchell, M.S., Matsuzaki, S., Imai, S., and Rao, V.B. (2002). Sequence analysis of bacteriophage T4 DNA packaging/terminase genes 16 and 17 reveals a common ATPase center in the large subunit of viral terminases. Nucleic Acids Res. 30, 4009–4021. Morais, M.C., Tao, Y., Olson, N.H., Grimes, S., Jardine, P.J., Anderson, D.L., Baker, T.S., and Rossmann, M.G. (2001). Cryoelectron-microscopy image reconstruction of symmetry mismatches in bacteriophage f29. J. Struct. Biol. 135, 38–46. Morais, M.C., Fisher, M., Kanamaru, S., Przybyla, L., Burgner, J., Fane, B.A., and Rossmann, M.G. (2004). Conformational switching by the scaffolding protein D directs the assembly of bacteriophage fX174. Mol. Cell 15, 991–997. Newcomb, W.W., Homa, F.L., and Brown, J.C. (2005). Involvement of the portal at an early step in herpes simplex virus capsid assembly. J. Virol. 79, 10540–10546. Orlova, E.V., Gowen, B., Dro¨ge, A., Stiege, A., Weise, F., Lurz, R., van Heel, M., and Tavares, P. (2003). Structure of a viral DNA gatekeeper at 10 A˚ resolution by cryo-electron microscopy. EMBO J. 22, 1255–1262. Otwinowski, Z., and Minor, W. (1997). Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326. Powell, D., Franklin, J., Arisaka, F., and Mosig, G. (1990). Bacteriophage T4 DNA packaging genes 16 and 17. Nucleic Acids Res. 18, 4005. Rao, V.B., and Black, L.W. (1988). Cloning, overexpression and purification of the terminase proteins gp16 and gp17 of bacteriophage T4. Construction of a defined in-vitro DNA packaging system using purified terminase proteins. J. Mol. Biol. 200, 475–488.

Gorbalenya, A.E., and Koonin, E.V. (1993). Helicases: amino acid sequence comparisons and structure-function relationships. Curr. Opin. Struct. Biol. 3, 419–429.

Rentas, F.J., and Rao, V.B. (2003). Defining the bacteriophage T4 DNA packaging machine: Evidence for a C-terminal DNA cleavage domain in the large terminase/packaging protein gp17. J. Mol. Biol. 334, 37–52.

Hendrix, R.W. (1998). Bacteriophage DNA packaging: RNA gears in a DNA transport machine. Cell 94, 147–150.

Rossmann, M.G., Moras, D., and Olsen, K.W. (1974). Chemical and biological evolution of nucleotide-binding protein. Nature 250, 194–199.

Jiang, W., Chang, J., Jakana, J., Weigele, P., King, J., and Chiu, W. (2006). Structure of epsilon15 bacteriophage reveals genome organization and DNA packaging/injection apparatus. Nature 439, 612–616.

Simpson, A.A., Tao, Y., Leiman, P.G., Badasso, M.O., He, Y., Jardine, P.J., Olson, N.H., Morais, M.C., Grimes, S., Anderson, D.L., et al. (2000). Structure of the bacteriophage f29 DNA packaging motor. Nature 408, 745–750.

Jones, T.A., Zou, J.-Y., Cowan, S.W., and Kjeldgaard, M. (1991). Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119. Kanamaru, S., Kondabagil, K., Rossmann, M.G., and Rao, V.B. (2004). The functional domains of bacteriophage T4 terminase. J. Biol. Chem. 279, 40795–40801. Lander, G.C., Tang, L., Casjens, S.R., Gilcrease, E.B., Prevelige, P., Poliakov, A., Potter, C.S., Carragher, B., and Johnson, J.E. (2006). The structure of an infectious P22 virion shows the signal for headful DNA packaging. Science 312, 1791–1795. Leffers, G., and Rao, V.B. (2000). Biochemical characterization of an ATPase activity associated with the large packaging subunit gp17 from bacteriophage T4. J. Biol. Chem. 275, 37127–37136. Leiman, P.G., Chipman, P.R., Kostyuchenko, V.A., Mesyanzhinov, V.V., and Rossmann, M.G. (2004). Three-dimensional rearrangement of proteins in the tail of bacteriophage T4 on infection of its host. Cell 118, 419–429. Mancini, E.J., Kainov, D.E., Grimes, J.M., Tuma, R., Bamford, D.H., and Stuart, D.I. (2004). Atomic snapshots of an RNA packaging motor reveal conformational changes linking ATP hydrolysis to RNA translocation. Cell 118, 743–755. Mitchell, M.S., and Rao, V.B. (2006). Functional analysis of the bacteriophage T4 DNA-packaging ATPase motor. J. Biol. Chem. 281, 518– 527.

Singleton, M.R., Scaife, S., and Wigley, D.B. (2001). Structural analysis of DNA replication fork reversal by RecG. Cell 107, 79–89. Soultanas, P., Dillingham, M.S., Velankar, S.S., and Wigley, D.B. (1999). DNA binding mediates conformational changes and metal ion coordination in the active site of PcrA helicase. J. Mol. Biol. 290, 137–148. Streisinger, G., Emrich, J., and Stahl, M.M. (1967). Chromosome structure in phage T4, III. Terminal redundancy and length determination. Proc. Natl. Acad. Sci. USA 57, 292–295. Subramanya, H.S., Bird, L.E., Brannigan, J.A., and Wigley, D.B. (1996). Crystal structure of a DExx box DNA helicase. Nature 384, 379–383. Velankar, S.S., Soultanas, P., Dillingham, M.S., Subramanya, H.S., and Wigley, D.B. (1999). Crystal structures of complexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism. Cell 97, 75–84. Walker, J.E., Saraste, M., Runswick, M.J., and Gay, N.J. (1982). Distantly related sequences in the a- and b-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1, 945–951. Accession Numbers The coordinates and associated structure factors have been deposited with the Protein Data Bank (accession numbers 2O0H [ATP complex], 2O0J [ADP complex], and 2O0K [Apo]).

Molecular Cell 25, 943–949, March 23, 2007 ª2007 Elsevier Inc. 949