The role of scaffolding proteins in the assembly of the small, single-stranded DNA virus φX1741

Article No. jmbi.1999.2699 available online at http://www.idealibrary.com on

J. Mol. Biol. (1999) 288, 595±608

The Role of Scaffolding Proteins in the Assembly of the Small, Single-stranded DNA Virus f X174 Terje Dokland1, Ricardo A. Bernal1, April Burch2, Sergei Pletnev1 Bentley A. Fane2 and Michael G. Rossmann1* 1

Department of Biological Sciences, Purdue University West Lafayette IN 47907-1392, USA 2

Department of Veterinary Sciences and Microbiology University of Arizona, Tucson AZ 85721, USA

An empty precursor particle called the procapsid is formed during assembly of the single-stranded DNA bacteriophage fX174. Assembly of the fX174 procapsid requires the presence of the two scaffolding proteins, D and B, which are structural components of the procapsid, but are not found in the mature virion. The X-ray crystallographic structure of a Ê resolution. This ``closed'' procapsid particle has been determined to 3.5 A structure has an external scaffold made from 240 copies of protein D, 60 copies of the internally located B protein, and contains 60 copies of each of the viral structural proteins F and G, which comprise the shell and the 5-fold spikes, respectively. The F capsid protein has a similar conformation to that seen in the mature virion, and differs from the previously Ê resolution electron microscopic reconstruction of the determined 25 A ``open'' procapsid, in which the F protein has a different conformation. The D scaffolding protein has a predominantly a-helical fold and displays remarkable conformational variability. We report here an improved and re®ned structure of the closed procapsid and describe in some detail the differences between the four independent D scaffolding proteins per icosahedral asymmetric unit, as well as their interaction with the F capsid protein. We re-analyze and correct the comparison of the closed procapsid with the previously determined cryo-electron microscopic image reconstruction of the open procapsid and discuss the major structural rearrangements that must occur during assembly. A model is proposed in which the D proteins direct the assembly process by sequential binding and conformational switching. # 1999 Academic Press

*Corresponding author

Keywords: Bacteriophage fX174; three-dimensional structure; procapsid; scaffolding proteins; morphogenesis

Introduction During morphogenesis, many viruses employ scaffolding proteins that mediate the assembly of the structural components into mature and infectious particles. These proteins are transiently associated with nascent protein complexes during virion assembly (King et al., 1980; Casjens & Hendrix, 1988; Kellenberger, 1990). In the absence of functional scaffolding proteins, erroneous and abortive assemblies often accumulate (Siden & Hayashi, 1974; Casjens & Hendrix, 1988). The Present address: T. Dokland, Institute of Molecular Agrobiology, National University of Singapore, Singapore 117604. E-mail address of the corresponding author: [email protected] 0022-2836/99/190595±14 $30.00/0

assembly process is guided by conformational switches induced when a capsid protein binds to another protein, such as a scaffolding protein. These structural changes alter binding surfaces, causing the next assembly step to differ from the previous one in the pathway. The different conformational states provide the energy to drive the assembly process forward (Steven, 1993). Conformational switching has been observed in the assembly of many icosahedral viruses, (King et al., 1980; Rossmann & Johnson, 1989), tailed-bacteriophages (Kellenberger, 1976; King et al., 1980; Dokland & Murialdo, 1993; Prasad et al., 1993), bacterial ¯agella (Asakura, 1970; Oosawa et al., 1994), microtubules (Burns & Symmons, 1995; Chretien et al., 1995) and actin ®laments (McGough, 1998). # 1999 Academic Press

596

Scaffolding Proteins of X174

One of the most de®ned macromolecular assemÊ bly systems is bacteriophage fX174. The 280 A diameter icosahedral capsid contains the four proteins F, G, H and J (Hall et al., 1959; Sinsheimer, 1959; Burgess, 1969; Table 1), enclosing a genome of positive sense, single-stranded (ss) DNA of 5386 nucleotides (Sanger et al., 1978). Sixty copies of protein F form the main shell of the virus, while 60 copies of protein G comprise the protruding ``spikes'' on the icosahedral 5-fold axes, giving the virion its characteristic shape (Burgess, 1969; McKenna et al., 1992). In addition, the virion contains 60 copies of the DNA-associated J protein and about 12 copies of the ``pilot'' protein H. Both the F and G proteins have the eight-stranded, antiparallel b-barrel motif common to many icosahedral plant and animal viruses (Rossmann & Johnson, 1989; Harrison et al., 1996). The ®rst step in fX174 assembly is the formation of F and G protein pentamers, the 9 S and 6 S particles, respectively (Tonegawa & Hayashi, 1970). A precursor capsid, called the procapsid or 108 S particle, is formed in the presence of the two scaffolding proteins, B and D (Fujisawa & Hayashi, 1977a; Mukai et al., 1979). In the procapsid, 240 copies of the scaffolding protein D form an external shell, while 60 copies of protein B occupy an internal location (Mukai et al., 1979; Ilag et al., 1995; Dokland et al., 1997). DNA packaging is accompanied by the gain of the DNA-binding protein J (Aoyama et al., 1981) and the loss of the B scaffolding protein (Fujisawa & Hayashi, 1977b), yielding the provirion, or 132 S particle (Weisbeek & Sinsheimer, 1974; Fujisawa & Hayashi, 1977b). Subsequent removal of the external scaffold yields the mature, infectious virion. Recently, we reported the structure of a scaffoldcontaining procapsid particle (Dokland et al., 1997, 1998). Comparisons with the procapsid electron microscopic (EM) reconstruction (Ilag et al., 1995) and the X-ray structure of the virion (McKenna et al., 1992) showed that this particle may have undergone maturation processes normally associated with DNA packaging and/or loss of the external scaffolding proteins during crystallization. Nevertheless, the closed procapsid retained a full complement of the two scaffolding proteins B and D. The maturation was shown to involve major conformational changes of the helical domain in the F capsid protein. These helices cover the 3-fold

axes in the mature virus and in the procapsid X-ray structure. The immature procapsid, represented by the EM reconstruction, has holes at the 3-fold axes big enough to permit ssDNA to enter the procapsid. Hence, the X-ray structure was termed the closed procapsid, while the EM reconstruction was referred to as the open procapsid. In both the closed and open procapsids, the D scaffolding proteins form a shell surrounding the F capsid. However, the genetic and EM data suggest that the immature open procapsid has a more intimate contact of the F capsid protein with the D scaffolding protein. The assembly process described here involves symmetry mismatches between the 240 D scaffolding proteins among themselves and with respect to the 60 F capsid proteins. The structure discussed shows a complete departure from a classical T ˆ 4 Caspar and Klug triangulation lattice (Caspar & Klug, 1962). It is becoming increasingly apparent that such symmetry adaptations are essential for the assembly of complex macromolecular machines and viruses and, indeed, are the rule rather than the exception for virus structures. Some examples are the ``T ˆ 2'' inner core of bacteriophage f6 and bluetongue virus, and its match with the outer T ˆ 13 shell (Butcher et al., 1997; Grimes et al., 1998). Similarly, the formation of erythropoietin (Livnah et al., 1999) dimers, for instance, lacks the usual oligomeric symmetry expected for the association of identical monomers. The structure of the closed procapsid has now been improved by inclusion of more data and crystallographic re®nement to give a ®nal R-factor of 27.3 %. The resultant structure, which shows some additional features, has been analyzed in terms of the differences between the four independent D scaffolding proteins and their contacts with the F capsid proteins. The improved structure was used to re-examine the conformational changes that must occur in going from the open to closed procapsid structure. We then describe the fX174 assembly pathway in terms of the three available structures: the EM reconstruction of the open procapsid (Ilag et al., 1995); the newly re®ned structure of the closed procapsid (this work); and the X-ray structure of the virion (McKenna et al., 1992), as well as newly available genetic and biochemical data (Burch et al., 1999).

Table 1. Structural proteins of fX174 Protein

Amino acids

M (kDa)

No. copies per particle

Virion proteins F G H J

426 175 328 37

48.4 19.0 34.4 4.2

60 60 12 60

Additional procapsid proteins D B

152 120

16.9 13.8

240 60

Location/function Shell/major capsid protein 5-Fold axes/major spike protein Internal/DNA pilot protein Internal/DNA binding protein External/scaffolding protein Internal/scaffolding protein

597

Scaffolding Proteins of X174

Results Structure determination The X-ray crystallographic structure of a fX174 Ê resolution by molprocapsid was solved to 3.5 A ecular replacement using a model based on the preÊ resolution cryo-EM viously determined 25 A reconstruction (Ilag et al., 1995) as a starting point for the phase determination. The details of the structure determination have been described elsewhere (Dokland et al., 1998). The ®nal R-factor agreement between the observed structure factor amplitudes and those derived from the Fourier back-transformed averaged map was 18.4 %, while the ``crystallographic'' R-factor between the observed structure factors and those calculated from the atomic model was 27.3 %. This rather high crystallographic R-factor is consistent with the poor quality and perhaps also because of the incompleteness of the data in the higher-resolution ranges. Nevertheless, due to the high non-crystallographic redundancy, the electron density map was readily interpretable, making it possible to build most of the amino acids of the D, F and G proteins, and part of the B protein. The model was partially re®ned using the program X-PLOR (BruÈnger, 1992; Table 2). The capsid protein The F capsid protein of fX174 contains an eightstranded, antiparallel b-barrel with its strands running roughly tangential to the shell surface. The b-barrel has two large insertions, the EF loop (residues 72-233) and the HI loop (residues 291-400), which make most of the contacts between adjacent subunits (McKenna et al., 1992; Dokland et al.,

1997), similar to the situation in the single-stranded DNA parvoviruses (Tsao et al., 1991). The b-barrel and associated small insertions of the F protein in the closed procapsid have a similar structure (r.m.s. deviation between 88 equivalenced Ca atoms is Ê ) to that found in the virion (Figure 1; Dokland 0.5 A et al., 1997). However, parts of the two large insertion loops (residues 166-232, 295-325 and 330-378) form an ``a-helical domain'' with larger (r.m.s. deviÊ ) conformational changes. The b-barrel ation of 2.0 A Ê relative to the domain is shifted by a total of 3.9 A virion structure, resulting in a rotation of 5.3  of the a-helical domain relative to the b-barrel domain (Tables 3 and 4). The 3-fold and 5-fold subunit interactions are retained, in spite of the overall movement of the protein. The greatest structural differences are found in residues 178-232 (r.m.s. Ê between Ca atoms). Residues 1-9 deviation of 2.9 A and 422-426, close to the 3-fold axes, are disordered in the closed procapsid structure. The spike protein The core structure of the G spike protein is an eight-stranded, antiparallel b-barrel whose strands run approximately perpendicular to the shell surface. Unlike protein F, the G protein has no large loops or other elaborations apart from 10 and 11 amino acid residue extensions found at the amino and carboxy terminus, respectively (McKenna et al., 1992). No signi®cant conformational difference can be detected between the G proteins in the virion Ê resolution. In and in the closed procapsid at 3.5 A the virion, the only contacts between the F and G proteins are ®ve polar and ®ve water-mediated interactions per G monomer (McKenna et al., 1994).

Table 2. Crystallographic re®nement of the procapsid A. Residues included in refinement Protein F G D1 D2 D3 D4 B

Range of ordered residues 4-421 1-175 6-148 6-138 5-144 7-152 1-8, 61-120

B. Final R-factor

27.3 (%)

C. Standard deviation from idealized values Bonds Angles Dihedral Improper

Ê) 0.016 (A 2.15 (deg.) 27.86 (deg.) 1.09 (deg.)

D. Average B-factor

E. Ramachandran plot statistics (%) Residues in most favored regions Residues in additional allowed regions Residues in generously allowed regions Residues in disallowed regions

Ê 2) 25.2 (A Individual B-factors range from 2 to 99

76.0 21.9 1.6 0.5

598

Scaffolding Proteins of X174

Figure 1. Stereo representation of the helical domain of the Ca backbone of the F protein in the procapsid structure (red) and the F protein in the virion (black). The superimposed b-barrel domain is colored green in the procapsid structure and blue in the virion. Symmetry axes are those in the virion.

In the closed procapsid, the b-barrel domain of the Ê further outwards, while F protein is situated 3.1 A Ê further out (Table 4). the G protein spikes are 5.5 A Therefore, the distance between the pentameric spikes and the underlying capsid shell is greater in the procapsid than in the virion. The G pentamers are bound to the capsid only through a few contacts with the external D scaffolding proteins (Table 5) and possibly also through the disordered terminal residues of the G protein. The external scaffolding protein The main difference in the electron density maps between the virion and procapsid is the presence of additional density in the procapsid map due the external D protein lattice. Each icosahedral asymmetric unit contains four copies (D1 through D4) of the external scaffolding protein (Figures 2 and Ê between the 3). There is a large gap of about 10 A shell formed by the external D scaffolding proteins and the F capsid protein. The contacts between the D and F shells are quite tenuous (Figure 4). The D protein has a predominantly a-helical fold and contains seven major helical stretches (helices 1-7; Figure 5), interspersed by short loop regions (loops 1-6), one of which (loop 5) also includes some b structure. The four copies of protein D per icosahedral asymmetric unit have different environments and, therefore, can be expected to have somewhat different conformations. Such differences would be minimized if the subunits were placed in quasiequivalent positions (Caspar & Klug, 1962; Caspar,

1980), but this is not the case in the fX174 procapsid. The environments of the four D subunits are completely different. The D1 and D3 subunits have almost identical conformations, while both D2 and D4 differ from this structural motif; D4 is the most structurally unique of the four subunits (Figure 6). There is a structurally conserved core of about 50 % of the sequence, corresponding to residues 27-60, 73-98 Ê and 116-128, among which there is a less than 2.5 A r.m.s. divergence between superimposed Ca atoms for any pair of D subunits (Table 3). The four subunits exist as two asymmetric dimers, where D1 and D2 form the ®rst dimer, and D3 and D4 the second dimer. There is an 51  rotation between the two subunits within each dimer. The two asymmetric dimers can be superimposed with a r.m.s. Ê for deviation between Ca atoms of less than 2.5 A 67 % of the residues. The contacts between the monomers within dimers are similar (Table 5). All Pro and Gly residues are in loops, except Gly61, which forms a kink in helix a3. Helices a2 and a3 (the N-terminal part) are highly charged, and helices a4 and a6 are strongly hydrophobic. The amino-terminal 23 residues of the D protein form the amphipathic helix 1, which in D1 and D2 is exposed to the exterior of the particle. In D3 and D4, this helix points towards the F protein. In D4, it is bent at Gln22 by 100  relative to its orientation in D1, D2 and D3. There are two cold-sensitive mutations in this helix, Gln12 ! Trp and Gln22 ! Tyr that confer a ``fragile procapsid'' phenotype, in which the procapsid disintegrates

Table 3. Difference between equivalent Ca Atoms Protein pair D1-D3 D2-D4 D1-D2 D3-D4 D1-D4 D2-D3 FCP-FMV all FCP-FMV a-helical domain FCP-FMV b-barrel domain GCP-GMV all

Total no. equivalencies 136 105 112 98 99 104 412 75 94 175

Êa No. Ca < 2.0 A 120 90 84 81 73 72 364 47 93 175

Ê) r.m.s. Difference (A equiv. res. 1.3 1.7 2.1 2.3 2.4 2.5 1.5 2.0 0.6 0.3

CP, closed procapsid; MV, mature virion. Ê after a least-squares ®t of equivalenced Ca atoms. The number of Ca atoms separated by less than 2 A

a

Rotational relationship (deg.) 115.3 111.6 52.8 49.3 165.6 65.0 5.3 2.4 5.3 0.2

599

Scaffolding Proteins of X174 Table 4. Radial outward shifts of proteins Closed P.C. (X-ray)open P.C. (EM)

Comparison: Protein F (b-barrel) G D1 D2 D3 D4

5.8a 6.0 5.5 5.3 4.0 3.9

Virion (X-ray)closed P.C. (X-ray)

Virion (X-ray)open P.C. (EM)

3.1 5.6 -

8.5 11.5 -

Ê. All values are in A Major conformational difference in addition to shift.

a

Table 5. Scaffolding protein contacts

Protein 1 D1

D2

D3

D2

D4

D1 D1 D2 D4

B

Secondary structure (l, loop; a, helix) a1 l1 a3 l3 a4 a50 a3 l3 a4 l5 a3 l3 a4 a50 a3 l3 l5 a5 l5 l 5, a6 a3, l 3, l 5

l5 a7

Residues 78, 94-97, 105-107 79, 109, 111, 113, 114 99-101 117 119 120 a

Protein 2 D2

D3

D4

D42a

D22a

G F F

F

Secondary structure (l, loop; a, helix) a5, a6 l 2, l 4 l 2, a5 l 2, a3, a5, a6 a5, a6 a6 l 2, a3, a5, l 5,

a3 a5 a6 a6

l 4, a5 a5, a6 a5, a6 a6 l5 l5 l5 l3 l 3, a3 Residues 134-136 Residues 161, 297, 338, 340, 388 Residue 150 Residues 89, 118, 183, 184, 191, 194, 197, 305, 307, 309, 311, 312, 324, 325, 326, 347 bG bF bB a3-a4 a2 a2, bH, bE

Subscri8pts indicate 2-fold-related neighboring subunits.

Type of contact Intra-dimer

Inter-dimer

Intra-dimer

2-fold

2-fold

Spike Capsid Capsid

Capsid

No. of atoms Protein 1/Protein 2 (polar) 4/5 (0) 5/3 (0) 5/7 (2) 16/27 (4) 3/5 (0) 11/12 (3) 7/8 (1) 17/20 (5) 3/3 (0) 9/9 (3) 6/7 (1) 13/19 (0) 4/5 (0) 7/13 (0) 18/13 (4) 7/5 (2) 4/4 (3) 1/1 (0) 19/30 (9) 8/8 (0) 9/7 (6)

1/1 (0) 21/33 (20)

84/77 (22)

Contact Ê 2) area (A 1309

933

1248

665

665

286 1342 260 1366

1451

Characteristics Hydrophobic Hydrophobic Ambivalent Hydrophobic Hydrophobic Hydrophobic Hydrophobic Hydrophobic Hydrophobic Ambivalent Hydrophobic Hydrophobic Hydrophobic Hydrophobic Hydrophobic Ambivalent Polar Hydrophobic Ambivalent Hydrophobic Polar

Hydrophobic Polar

Hydrophobic

600

Scaffolding Proteins of X174

Figure 2. Surface representation calculated from the coordinates at Ê resolution of the closed pro10 A capsid (left) and virion (right) structures. The asymmetric triangle is indicated on both structures. The different proteins subunits in the icosahedral asymmetric unit and the neighboring subunits are labeled F, G, D1, D2, D3 and D4. Subscripts indicate 2-fold- and 5-fold-related neighboring subunits.

during DNA packaging (Fane et al., 1993; Ekechukwu & Fane, 1995). The effects of these mutations can be suppressed by a number of amino acid substitutions clustered in helices 4, 5 and 8 of the F protein. The suppressor mutations within helix 5 of the F protein are at contact points across 2-fold axes, suggesting that strengthening contacts between pentamers can compensate for weakened scaffolding-capsid interactions. The mutations within helices 4 and 8 of the F protein and the closed procapsid structure may indicate that there is an interaction between the F protein and helix 1 of the D3 or D4 subunit at some time during assembly (see below). Residues 47-66 of the D protein form helix 3, the ®rst part of which is rich in arginine residues. Although the ®rst 13 amino acid residues of this helix belong to the structurally conserved core, there is a striking conformational difference between subunits in the second part of this helix, continuing into loop 3. In D1 and D3, helix 3 is bent by 82  at Gly61 and loop 3 makes contact

Figure 3. Superposition of virion structure onto proÊ resolution. capsid electron density at 6.5 A

between the two monomers within each asymmetric dimer (Figure 5). In D2 and D4, this helix is straight and loop 3 is in a different conformation. This may prevent further binding of D proteins via the same interaction to form a helical oligomer. Loop 3 is also unusually rich in proline residues which along with Gly61 may be responsible for the ¯exibility and hinge movement observed in this loop. This could facilitate the large structural changes in the loop, presumably induced on formation of a dimer. Helix 3 is also involved in contacts between 2-fold-related D3 subunits through residue Arg52 in one subunit with Glu56 and Asp64 in the other subunit. Loop 5 and helix 6 of subunit D1 make the few contacts that exist between the scaffold and spike proteins (Table 5). This tenuous connection is all that appears to hold the spikes to the underlying capsid shell in the procapsid. Helix 4 is hydrophobic and is almost completely buried (Figure 5). Helix 4, loop 4, helix 5 and helix 6 are all in the structurally conserved core of the D subunit and make many contacts within the scaffold, both within and between dimers (Table 5; Figure 7). Loop 5 forms a hairpin turn and is structurally polymorphic among the four subunits. This loop forms an a-helix, a b-strand, or a loop structure. In D1 and D3, loop 5 forms a small helix (a5') near its tip, which is absent in D2 and D4 where it forms an antiparallel b-ribbon. The major role of loop 5 appears to be the formation of contacts across the 2-fold axis. This function is carried out almost exclusively by two 2-fold-related D3 subunits in which loop 5 has a unique conformation (Figure 5). In spite of its role in D3, the loop is partly disordered in that subunit. Like the kink in helix a3, loop 5 presumably acts as a conformational switch during assembly, allowing these 2-fold contacts to form only after the formation and assembly of the asymmetric dimer. The carboxy terminus of the D protein (residues 130-152) contains many charged residues and is highly variable in conformation. In D1, D2 and D3, it is exposed to the solvent and is either completely or partly disordered (Figure 5). It does not make any contact, except in the D4 subunit, where it is

601

Scaffolding Proteins of X174

Figure 4. ``Roadmap'' plot of the surface residues of the F protein, showing the residues that make contact with the four copies of protein D (D1, green; D2, yellow; D3, red; D4, blue).

ordered all the way to residue 152, forming a long, straight helix. This helix is responsible for the predominant interactions between the external scaffold and the capsid protein. The direction of helix 7 in D4 is 61  relative to the direction of the partly disordered amino-terminal arm in D1. Most of the contacts made between the D scaffolding proteins are within the same geometric asymmetric unit. There are relatively few contacts generated by the icosahedral 2-fold axes (Table 5), although these are primarily responsible for connecting adjacent pentameric shell units. Formation of these contacts may be the primary function of the D scaffolding protein, as the EM image reconstruction of the open procapsid shows little contact between the F proteins across the 2-fold axes. Without these contacts, observed in both the EM reconstruction of the open procapsid and in the X-ray structure of the closed procapsid, the procapsid would most likely be unstable. That the D proteins may be the primary agents for forming an icosahedral capsid is supported by the formation of 20 S particles, which could possibly be spherical scaffolding-related particles containing a high concentration of D protein (Farber, 1976).

The internal scaffolding protein The amino-terminal residues (1 to 8) and carboxy-terminal residues (61 to 120) of the B scaffolding protein have been identi®ed in the closed procapsid electron density map. The previously reported structure (Dokland et al., 1997) reported only residues 1 to 8 and 80 to 120. Absence of ordered density for residues 9 to 60 might imply a ¯exible character for the central section of the B protein, or the B protein might be cleaved in an autocatalytic process (unpublished results) required in the assembly pathway. The B protein makes extensive hydrophobic contacts with the b-barrel on the internal surface of the F protein. The carboxy-terminal residues bind to the inside of the capsid protein at essentially the same site as the DNA binding protein J in the mature virion (Figure 8). Since the B protein is extruded from the procapsid during DNA packaging, elimination of the B protein may be due to its competition by the J protein and the negatively charged DNA for the binding site on F. Although the structures of the B and J proteins are rather different, their interactions with the F protein are very similar (Table 6, Figure 8). Residues 117, 119 and 120 of B are equivalent to residues 35, 36 and 37 of the J protein. There is a hydrophobic pocket on the internal surface of the F

602

Scaffolding Proteins of X174 Figure 5. Sequence and structural elements of the D proteins. The secondary structure elements are indicated as spirals (a helix) and zigzag lines (b sheet). Loop regions are shown as dotted lines. The asterisks (*) indicate the kink in helix 3 of the D1 and D3 subunits. The yellow-highlighted parts of the sequence denote the structurally conserved core regions of 75 resiÊ ). Residues (r.m.s. deviation <2.5 A dues 103-114 of loop 5 in the D4 subunit are poorly ordered and could not be built with con®dence. A few N-terminal residues, as well as part of the C terminus in some subunits (most notably D2), were not seen in the map and are left blank in the alignment. Mutations in residues Gln12 ! Trp and Gln22 ! Tyr (boxed) cause a ``fragile procapsid'' phenotype (Ekechukwu & Fane, 1995). Residues that make contact with other subunits are shown here and summarized in Table 5.

Figure 6. Superposition of the Ca backbone of D1 (green) onto (a) D2 (orange) and onto (b) D4 (blue).

Scaffolding Proteins of X174

603

Figure 7. Ribbon representation of the closed procapsid proteins contained within two 2-fold-related asymmetric units. The interior of the particle is at the bottom, while the exterior is at the top of the page. Color code: D1, green; D2, yellow; D3, red; D4, blue; F, purple; G, pink; B, orange.

protein consisting of Phe67 in strand E of the b-barrel as well as Tyr134 and Phe135 in helix 2 of the F protein, which binds Phe120 of the B protein or Phe37 of the J protein. There are negative charges on Glu98, Asp105, Glu111, and Asp113 of the B protein that interact with Lys166, Arg239, and Arg274 on the F protein, but there is no equivalent interaction between the J and F proteins (Table 6). Presumably, the negative charge on the DNA associated with the J protein helps to displace the B protein. There is also equivalence in the formation of hydrogen bonds between the B and F proteins and between the J and F proteins. Similar

interactions were observed for the B protein in the structure of the degraded procapsid of the related virus, bacteriophage G4 (McKenna et al., 1996; and unpublished results). Genetic analyses underscore the importance of the interactions within the carboxy-terminal region of the B protein (Burch et al., 1999). If the B protein of bacteriophage G4 is replaced by the B protein of fX174, infectious particles are formed only when the F protein of G4 has a mutation (Ser140 ! Phe) in the region where it is in contact with the B protein. The results of genetic analyses also suggest that B protein binding to the internal surface of the F protein induces

Figure 8. Stereo diagram showing the residues that make contacts between protein F (purple) and the carboxyterminal part of protein B (orange). In green is shown the carboxy-terminal part of the DNA-binding protein J from the virion structure, after shifting it by the same amount that the b-barrel in the virion is shifted relative to that in the procapsid. Note the almost perfect overlap between proteins B and J in the part nearest protein F, suggesting a competition between the same binding site.

604

Scaffolding Proteins of X174

Table 6. Charge interaction of the B and F proteins B Protein Asp98 Glu105 Asp111 Asp111 Glu113 Glu113 Glu113

O OE2 OD1 OD2 OE1 OE2 OE2

F Protein Arg274 Arg274 Arg239 Arg239 Lys239 Arg239 Arg239

NH1 NH1 NH1 NH1 NZ NH1 NH2

Ê) Distance (A 2.92 3.35 3.51 2.91 2.57 3.01 3.57

conformational changes on the external surface. A cold-sensitive mutation in fX174 (Gln20 ! Ser) blocks the association of 9 S and 6 S particles at non-permissive temperatures. This phenotype can be suppressed by a number of mutations on the external surface of the F protein. The absence of this B protein-mediated conformational switch can also lead to the premature association of 9 S particles that self-assemble into irregular 55 to 65 S structures (Siden & Hayashi, 1974). The function of the B protein might be to alter the 9 S pentameric intermediate to an assemblycompetent unit (Siden & Hayashi, 1974; Fane & Hayashi, 1991; Ekechukwu & Fane, 1995; Burch et al., 1999). The cryo-EM image reconstruction (Ilag et al., 1995) and X-ray structure suggest that B proteins participate in dimer formation, consistent with the need for the B protein in assembling 9 S pentamers into icosahedral procapsids. Thus, the B and D proteins might have similar internal and external tethering functions in bridging 2-fold related capsid protein pentamers. The H pilot protein There is extensive protein-like density inside the capsid in the vicinity of the 5-fold axes. This density is at about a quarter of the maximum level of the F and G proteins. It is likely that this density represents the H protein, which acts as the ``pilot protein'' that is ejected through a 5-fold spike together with the DNA upon infection (Jazwinski et al., 1975; Yazaki, 1981). The association of this density with the H protein is consistent with the cryo-EM reconstructions of the procapsid (Ilag et al., 1995) and virion (Olson et al., 1992), as well as with biochemical data, which associates one H protein with each pentameric spike (Shank et al., 1977). The H protein density would be an average of ®ve different orientations, on account of the icosahedral averaging of the map. Comparison between EM and X-ray structures The observed similarity between the capsid protein in the X-ray structures of the virion and procapsid was surprising, since the corresponding EM structures had displayed major differences in the F protein shell (Ilag et al., 1995). In the EM reconstruction of the procapsid, the F protein b-barrel domain appeared to be rotated by  77  with respect to the virion. In addition, the two major insertion loops

seemed to be in radically different conformations, which accounted for the large hole at the 3-fold axes of the procapsid where three helices formed a protruding knob in the virion. If the EM structure represents the true procapsid intermediate, then the similarity between the two X-ray structures is due to a spontaneous conformational change of the metastable procapsid. Such spontaneous maturation is commonly seen in viral procapsids (Dokland & Murialdo, 1993; Prasad et al., 1993; Zhou et al., 1998), and may be induced by high salt, as in the procapsid crystallization conditions, chaotropic agents like urea, or even prolonged storage. A similar situation occurred for bacteriophage G4, a virus homologous to fX174, where the crystallization had induced both conformational change in the capsid protein and loss of the external scaffold, resulting in a structure similar to that of the mature virion, albeit devoid of DNA (McKenna et al., 1996). On the other hand, the previous model building into the EM electron density suffered from a number of inaccuracies. One problem was the lack of any structural information on the D protein, and the false conclusion that there were only 120 copies of this protein in the procapsid. Furthermore, due Ê ) resolution of the EM reconstructo the low (25 A tion, the wrong hand was chosen for the EM structure, resulting in an incorrect ®t for the F protein into the shell region density. Nevertheless, the locations of the two scaffolding proteins B and D were correctly identi®ed. With the current knowledge of the structure and stoichiometry of the D protein, as well as the absolute hand of the map, a revised model was built into the EM reconstruction of the open procapsid. The assumption was made that the b-barrel domain would move only minimally, while parts of the loops could be allowed extensive movement and conformational change. The G and D proteins from the closed procapsid X-ray structure could readily be brought into the open procapsid EM density by Ê and 5.6 A Ê , respectively, in a simple shifts of 6.2 A predominantly outwards radial direction. There is thus a continuum of radial adjustment of these proteins from the open procapsid through the closed procapsid to the virion, represented by a shrinkage or collapse of the capsid surrounding the DNA (Table 4). However, when a similar radial shift is applied to the F protein, major portions of the protein are outside the density. The most striking differences between the open and closed forms of the procapsid occur at the 2-fold and 3-fold axes, the sites of extensive interactions between adjacent protein F pentamers in the virion and in the closed proÊ diameter capsid. The open procapsid has a 30 A hole at the 3-fold axes (Figure 9). The residues that fall outside the density include residues 175-231, representing part of the a-helical domain. The small conformational differences between the virion and the closed procapsid, represented by a small angular change between the b-barrel and a-helical domains, could be a relic of a larger difference found at an earlier stage of assembly. The B and J binding surfaces

Scaffolding Proteins of X174

605

Figure 9. View down the 3-fold axis showing the relationship of the F protein as seen in the closed procapsid when superimposed onto the EM density of the open procapsid. The big hole on the 3-fold axis in the EM reconstruction of the open procapsid is covered by helix 4 of the F protein in the closed procapsid X-ray structure. Gray and Ê resolution EM structure. blue contours represent two density levels in the 25 A

to F include the residues 175-231 in the F protein, which must undergo the major conformational changes during or after DNA packaging. f X174 assembly pathway The ®rst detectable intermediates in the fX174 assembly pathway are the 9 S and 6 S pentameric particles (Tonegawa & Hayashi, 1970) of the capsid and spike proteins, respectively. These particles are seen transiently in wild-type infected cells. In cells infected with temperature-sensitive or cold-sensitive mutants of the internal scaffolding protein, 9 S and 6 S particles accumulate at restrictive temperatures. These particles can be chased into larger structures following a temperature shift (Siden & Hayashi, 1974; Ekechukwu & Fane, 1995). In the

absence of D proteins, at least three structurally distinct 12 S particles have been isolated (Tonegawa & Hayashi, 1970; Hayashi et al., 1988; Fane & Hayashi, 1991; Ekechukwu & Fane, 1995). All three contain the F and G proteins, but differ in the incorporation of the H and B proteins, as well as their ability to be assembled into larger particles. Some of these 12 S particles exhibit the biochemical properties traditionally associated with morphogenetic intermediates; namely, the ability to be chased into large particles in temperature-shift experiments. However, it is not clear whether these particles represent a true intermediate or the product of an off-pathway reaction (Hayashi et al., 1988). With the available structural, genetic and biochemical data, it is dif®cult to visualize how the

606 12 S particle could be a true intermediate in the assembly of the open procapsid. We, therefore, speculate that there is an intermediate similar to the 12 S particle, but also containing 20 D proteins. Such a particle would readily lose the D proteins, degenerating into a 12 S-like particle, thus explaining why this postulated intermediate has never been isolated. The existence of the same dimeric subassembly units D1:D2 and D3:D4 in two completely different environments implies that the dimer may be a basic requirement for the assembly of this intermediate. The open procapsid is then assembled from this postulated intermediate. The principal contacts in the closed procapsid between the 6 S pentameric G proteins and the D scaffolding proteins involve helix 7 of D1. Secondsite genetic analyses, however, suggest that there must be additional contacts not seen in the closed procapsid structure. A fragile procapsid phenotype exhibited by two fX174 mutants has amino acid substitutions in helix 1 of the D scaffolding protein (Gln12 ! Trp and Gln21 ! Tyr). During DNA packaging, procapsids of these mutants dissociate into 12 S-like particles that have lost the ability to be chased into larger structures (Ekechukwu & Fane, 1995). These effects can be suppressed by mutations in helix 4 of the F capsid protein. Helix 4 of the F protein must be situated close to the 3fold and 2-fold axes in the open procapsid in order to create a hole at the 3-fold axis. This would also place helix 4 of F close to helix 1 of the D4 subunit. Such a structure is consistent with the abovedescribed model building into the cryo-EM density (Figure 9). Experiments with a chimeric D protein (unpublished results) between fX174 and the related phage alpha3 also suggest that there is an interaction between helix 1 of the D4 subunit and the capsid protein F during morphogenesis. The last stage of morphogenesis involves the concurrent synthesis and packaging of singlestranded (‡) viral genome. A pre-initiation complex consisting of the viral proteins A and C, replicative form DNA, and the host cell rep protein associates with the viral procapsid, forming the 50 S complex (Mukai et al., 1979; Ekechukwu et al., 1995). Mutant host rep proteins that speci®cally block this association have been isolated and characterized (Tessmann & Peterson, 1976; Ekechukwu et al., 1995). Mutations within the fX174 capsid protein restore the ability of the procapsid to associate with the pre-initiation complex. These mutations are in a depression within the protein that skirts the 2-fold axis of symmetry. This depression is still exposed in the structure of the closed procapsid and may delineate the binding site of the pre-initiation complex. Genome biosynthesis and packaging dislodges the internal scaffolding protein, yielding the 132 S particle (Fujisawa & Hayashi, 1977a,b). At the same time, closure of the 3-fold holes triggers the release of the F protein from the surrounding D lattice, leading to the collapse of the F protein into its mature form. But for the absence of the DNA, this is the

Scaffolding Proteins of X174

stage represented by the present closed procapsid structure. This collapse also enables the subsequent release of the D protein, which completes the maturation process (Fujisawa & Hayashi, 1977a,b).

Methods The methods used for puri®cation, crystallization, Xray crystallographic structure determination, and electron microscopy were described by Dokland et al. (1997, 1998). However, the original warm X-ray diffraction data plus some additional data were re-processed using the DENZO program (Otwinowski & Minor, 1997) and scaled with the SNP program (Bolotovsky et al., 1998; Table 7). Partial re¯ections greater than 0.3 were converted to their full equivalent and used for averaging. The data contained 30 crystals with a total of 65 frames. The oscillation angles were chosen as 0.20  , 0.25  , or 0.30  . Cell dimensions associated with each crystal were post-re®ned as independent parameters and then averaged to Ê . The previously determined phases 771.7(2.9) A Ê resolution in the (Dokland et al., 1998) were used to 3.5 A ®rst cycle of electron density averaging using the program ENVelope (Rossmann et al., 1992). Previously unobserved re¯ections were phased with calculated phases from the averaged map. Subsequently, scaled Fcalc amplitudes were used for unobserved re¯ections in map calculations. A total of ten averaging cycles were performed. The orientation and position of the two independent particles were checked using the ``climb'' procedure (Muckelbauer et al., 1995) in which the differences between the non-crystallographic symmetry (NCS)-related densities is minimized. The particle in position (ÿ0.005, ÿ0.005, ÿ0.005) moved by 0.03  , but no other change was detected. The ®nal correlation coef®cients are compared to earlier results in Table 8. The new data gives better overall results, particuÊ. larly in the range of 9.7 to 4.0 A The earlier model (Dokland et al., 1997, 1998) was rebuilt in accordance with the new map. A few sections in the D and F proteins required major alterations. Furthermore, electron density corresponding to residues 61 to 79 of the B protein was now visible and could be rebuilt. The new model was re®ned by conjugate gradient minimization using the program X-PLOR (BruÈnger, 1992), using the procedure as described by Dokland et al. (1998). The initial R-factor was 31.6 %. After 80 cycles of Powell minimization including the electrostatic potential Table 7. Data collection statistics for warm (4 C) reprocessed data Resolution 1-7.53 7.53-5.98 5.98-5.23 5.23-4.75 4.75-4.41 4.41-4.15 4.15-3.94 3.94-3.77 3.77-3.63 3.63-3.50 overall

Rmergea

Number of reflections

Completenessb

12.7 20.8 26.2 26.8 28.6 29.9 34.2 33.9 36.2 36.0 21.7

81,308 83,804 82,666 80,147 71,348 63,464 57,887 47,913 38,006 25,651 632,194

86.4 89.0 87.8 85.1 75.8 67.4 61.5 50.9 40.4 27.2 67.1

a Rmerge ˆ 100hij(Ihi ÿ hIhi)j/hiIhi; all ``un¯agged'' measured intensities were included. b Completeness is given for all re¯ections with I/s(I) > 3.

607

Scaffolding Proteins of X174 Table 8. Final correlation coef®cients as a function of resolution Resolution range

Previous results

1-19.91 19.91-12.33 12.33-9.70 9.70-8.25 8.25-7.30 7.30-6.62 6.62-6.10 6.10-5.69 5.69-5.35 5.35-5.06 5.06-4.81 4.81-4.61 4.61-4.42 4.42-4.25 4.25-4.11 4.11-3.97 3.97-3.85 3.85-3.74 3.74-3.64 3.64-3.54 Overall

0.924 0.939 0.939 0.933 0.924 0.912 0.896 0.888 0.889 0.882 0.860 0.820 0.793 0.766 0.741 0.669 0.576 0.587 0.611 0.631 0.868

Present results 0.870 0.927 0.927 0.938 0.933 0.927 0.914 0.910 0.908 0.903 0.892 0.863 0.842 0.816 0.792 0.727 0.564 0.511 0.528 0.528 0.873

terms, the R-factor had dropped to 28.0 % with reasonable geometry (Table 2). No Rfree was calculated, as the high NCS causes Rfree and RWorking to be almost identical (Silva & Rossmann, 1987; Had®eld et al., 1997). Finally, the independent but restrained isotropic temperature factors were re®ned in 20 cycles causing R to drop to 27.3 %. Protein Data Bank The coordinates have been deposited with the Protein Data Bank (accession number 1cd3).

Acknowledgments We are grateful to Sharon Wilder and Cheryl Towell for help in the presentation of the manuscript. T.D. was supported, in part, by a European Molecular Biology Organization postdoctoral fellowship. The work was funded by grants form the National Science Foundation to M.G.R. and B.A.F.

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Edited by I. A. Wilson (Received 14 December 1998; received in revised form 10 March 1999; accepted 10 March 1999)