HK97 Maturation Studied by Crystallography and H/H Exchange Reveals the Structural Basis for Exothermic Particle Transitions

HK97 is an exceptionally amenable system for characterizing major conformational changes associated with capsid maturation in double-stranded DNA bacteriophage. HK97 undergoes a capsid expansion of ∼ 20%, accompanied by major subunit rearrangements during genome packaging. A previous 3.44-Å-resolution crystal structure of the mature capsid Head II and cryo-electron microscopy studies of other intermediate expansion forms of HK97 suggested that, primarily, rigid-body movements facilitated the maturation process. We recently reported a 3.65-Å-resolution structure of the preexpanded particle form Prohead II (P-II) and found that the capsid subunits undergo significant refolding and twisting of the tertiary structure to accommodate expansion. The P-II study focused on major twisting motions in the P-domain and on refolding of the spine helix during the transition. Here we extend the crystallographic comparison between P-II and Head II, characterizing the refolding events occurring in each of the four major domains of the capsid subunit and their effect on quaternary structure stabilization. In addition, hydrogen/deuterium exchange, coupled to mass spectrometry, was used to characterize the structural dynamics of three distinct capsid intermediates: P-II, Expansion Intermediate, and the nearly mature Head I. Differences in the solvent accessibilities of the seven quasi-equivalent capsid subunits, attributed to differences in secondary and quaternary structures, were observed in P-II. Nearly all differences in solvent accessibility among subunits disappear after the first transition to Expansion Intermediate. We show that most of the refolding is coupled to this transformation, an event associated with the transition from asymmetric to symmetric hexamers.     

Crosslinking renders bacteriophage HK97 capsid maturation irreversible and effects an essential stabilization

In HK97 capsid maturation, structural change ('expansion') is accompanied by formation of covalent crosslinks, connecting residue K169 in the 'E-loop' of each subunit with N356 on another subunit. We show by complementation experiments with the K169Y mutant, which cannot crosslink, that crosslinking is an essential function. The precursor Prohead-II passes through three expansion intermediate (EI) states en route to the end state, Head-II. We investigated the effects of expansion and crosslinking on stability by differential scanning calorimetry of wild-type and K169Y capsids. After expansion, the denaturation temperature (Tp) of K169Y capsids is slightly reduced, indicating that their thermal stability is not enhanced, but crosslinking effects a major stabilization (deltaTp, +11 degrees C). EI-II is the earliest capsid to form crosslinks. Cryo-electron microscopy shows that for both wild-type and K169Y EI-II, most E-loops are in the 'up' position, 30 A from the nearest N356: thus, crosslinking in EI-II represents capture of mobile E-loops in 'down' positions. At pH 4, most K169Y capsids remain as EI-II, whereas wild-type capsids proceed to EI-III, suggesting that crosslink formation drives maturation by a Brownian ratchet mechanism.     

Evidence that a local refolding event triggers maturation of HK97 bacteriophage capsid

Bacteriophage capsids are a striking example of a robust yet dynamic genome delivery vehicle. Like most phages, HK97 undergoes a conformational maturation that converts a metastable Prohead into the mature Head state. In the case of HK97, maturation involves a significant expansion of the capsid and concomitant cross-linking of capsid subunits. The final state, termed Head-II, is a 600 angstroms diameter icosahedral structure with catenated subunit rings. Cryo-EM, small angle X-ray scattering (SAXS), and biochemical assays were used previously to characterize the initial (Prohead-II) and final states (Head-II) as well as four maturation intermediates. Here we extend the characterization of the acid-induced expansion of HK97 in vitro by monitoring changes in intrinsic fluorescence, circular dichroism (CD), and SAXS. We find that the greatest changes in all observables occur at an early stage of maturation. Upon acidification, fluorescence emissions from HK97 exhibit a blueshift and decrease in intensity. These spectral changes reveal two kinetic phases of the expansion reaction. The early phase exhibits sensitivity to pH, increasing in rate nearly 200-fold when acidification pH is lowered from 4.5 to 3.9. The second, slower phase reported by fluorescence is relatively insensitive to pH. Time-resolved SAXS experiments report an increase in overall particle dimension that parallels the fluorescence changes for the early phase. Native agarose gel assays corroborated this finding. By contrast, probes of CD at far-UV indicate that secondary structural changes precede the early expansion phase reported by SAXS and fluorescence. Based on the crystallographic structure of Head-II and the pseudo-atomic model of Prohead-II, we interpret these changes as reflecting the conversion of subunit N-terminal arms (N-arm) from unstructured polypeptide to the mixture of beta-strand and beta-turn observed in the Head-II crystal structure. Refolding of the N-arm may thus represent the conformational trigger that initiates the irreversible expansion of the phage capsid.     

Control of crosslinking by quaternary structure changes during bacteriophage HK97 maturation

Radical structural changes drive the maturation of the capsid of HK97, a lambda-like, dsDNA bacteriophage of Escherichia coli. These include expansion from approximately 560 to approximately 660 A in diameter, metamorphosis from a round to an angular shape, and formation of covalent crosslinks between adjacent capsomers. Analogous transformations also occur in unrelated viruses and protein complexes. We find that expansion and crosslinking happen concurrently during maturation at low pH. Expansion causes residues on three different subunits to move up to 35 A to form 420 active sites that each catalyze the formation of a lysine-asparagine crosslink between adjacent subunits, making crosslink formation an indirect reporter of structural change. Intermediate crosslinking patterns support a previously proposed model of expansion, while hydrophobic properties aid in distinguishing discrete intermediates. A structure derived from cryo-EM images reveals the free intermediate conformation of penton arms, supporting our model for coordinated movement of hexons and pentons on the capsid lattice.     

Capsid conformational sampling in HK97 maturation visualized by X-ray crystallography and cryo-EM

Maturation of the bacteriophage HK97 capsid from a precursor (Prohead II) to the mature state (Head II) involves a 60 A radial expansion. The mature particle is formed by 420 copies of the major capsid protein organized on a T = 7 laevo lattice with each subunit covalently crosslinked to two neighbors. Well-characterized pH 4 expansion intermediates make HK97 valuable for investigating quaternary structural dynamics. Here, we use X-ray crystallography and cryo-EM to demonstrate that in the final transition in maturation (requiring neutral pH), pentons in Expansion Intermediate IV (EI-IV) reversibly sample 14 A translations and 6 degrees rotations relative to a fixed hexon lattice. The limit of this trajectory corresponds to the Head II conformation that is secured at this extent only by the formation of the final class of covalent crosslinks. Mutants that cannot crosslink or EI-IV particles that have been rendered incapable of forming the final crosslink remain in the EI-IV state.     

Cooperative reorganization of a 420 subunit virus capsid

The complex protein capsids of many viruses exhibit dramatic reorganizations at critical stages in their life-cycle. Here, time-resolved solution X-ray scattering was used to study a dynamic, large-scale conformational maturation of the 420 subunit, 13 MDa, icosahedrally symmetric HK97 bacteriophage capsid. Isoscattering points in the time-resolved scattering patterns and singular value decomposition revealed that the expansion occurs as a cooperative, two-state reaction. The analysis demonstrates that the population shift from Prohead-II to Expansion Intermediate I, EI-I (60 A larger than Prohead-II) occurs in minutes, but does not reveal the time required for individual transitions that occur stochastically. Any intermediate forms that may be traversed during this conversion are unstable and do not constitute an appreciable population of the ensemble of particles. In an energetic landscape view, particles must undergo an energy barrier-crossing event in order to successfully convert from Prohead-II to EI-I. This implies that the particles "hop" over the energy barrier stochastically as they individually attain an expansion-active state. Interestingly, systematic deviations from single-exponential kinetics were observed for the population shift. This may indicate that in undergoing the irreversible conversion from Prohead-II to EI-I, particles are subject to a complex energy landscape that links the initial and final particle forms.     

Time-resolved molecular dynamics of bacteriophage HK97 capsid maturation interpreted by electron cryo-microscopy and X-ray crystallography

The bacteriophage HK97 capsid is a molecular machine that exhibits large-scale conformational rearrangements of its 420 identical protein subunits during capsid maturation. Immature empty capsids, termed Prohead II, assemble in vivo in an Escherichia coli expression system. Maturation of these particles may be induced in vitro, converting them into Head II capsids that are indistinguishable in conformation from the capsid of an infectious phage particle. One method of in vitro maturation requires acidification to drive the reaction through two expansion intermediates (EI-I, EI-II) to its penultimate particle state (EI-III), which has 86% more internal volume than Prohead II. Neutralization of EI-III produces the fully mature capsid, Head II. The three expansion intermediates and the acid expansion pathway were characterized by cryo-EM analysis and 3D reconstruction. We now report that, although large-scale structural changes are involved, the electron density maps for these intermediate states are readily interpreted in terms of quasi-atomic models based on subunit structures determined by prior crystallographic analysis of Head II. Progression through the expansion intermediate states primarily represents rigid-body rotations and translations of the subunits, accompanied by refolding of two small regions, the N-terminal arm and a beta-hairpin called the E-loop. Movies made with these pseudo-atomic coordinates and the Head II X-ray coordinates illuminate various aspects of the maturation pathway in the course of which the pattern of inter-subunit interactions is sequentially transformed while the integrity of the capsid is maintained.     

The refined structure of a protein catenane: the HK97 bacteriophage capsid at 3.44 A resolution

The HK97 bacteriophage capsid is a unique example of macromolecular catenanes: interlocked rings of covalently attached protein subunits. The chain mail organization of the subunits stabilizes a particle in which the maximum thickness of the protein shell is 18A and the maximum diameter is 550A. The electron density has the appearance of a balloon illustrating the extraordinary strength conferred by the unique subunit organization. The refined structure shows novel qualities of the HK97 shell protein, gp5 that, together with the protease gp4, guides the assembly and maturation of the virion. Although gp5 forms hexamers and pentamers and the subunits exist in different structural environments, the tertiary structures of the seven protein molecules in the viral asymmetric unit are closely similar. The interactions of the subunits in the shell are exceptionally complex with each subunit interacting with nine other subunits. The interactions of the N-terminus released after gp5 cleavage appear important for organization of the loops that become crosslinked to the core of a neighboring subunit at the maturation. A comparison with a model of the Prohead II structure revealed that the surfaces of non-covalent contact between the monomers that build up hexamers/pentamers are completely redefined during maturation.     

Maturation dynamics of bacteriophage HK97 capsid

Maturation of the bacteriophage HK97 capsid requires a large conformational change of the virus capsid. Experimental studies have identified several intermediates along this maturation pathway. To gain insights into the molecular mechanisms of capsid maturation, we examined the fluctuation dynamics of the procapsid and mature capsid using a residue-level computational approach. The most cooperative motions of the procapsid are found to be consistent with the observed change in configuration that takes place during maturation. A few dominant modes of motion are sufficient to describe the anisotropic expansion that accompanies maturation. Based upon these modes, maturation is proposed to occur via an overall expansion and reconfiguration of the capsid initiated by puckering of the pentamers, followed by flattening and crosslinking of the hexameric subunits, and finally crosslinking of the pentameric subunits. The highly mobile E loops are stabilized by anchoring to highly stable residues belonging to neighboring subunits.     

Domain structures and roles in bacteriophage HK97 capsid assembly and maturation

Head assembly in the double-stranded DNA coliphage HK97 involves initially the formation of the precursor shell Prohead I from approximately 420 copies of a 384-residue subunit. This is followed by proteolytic removal of residues 2-103 to create Prohead II, and then reorganization and expansion of the shell lattice and covalent cross-linking of subunits make Head II. Here, we report and structurally interpret solution Raman spectra of Prohead I, Prohead II, and Head II particles. The Raman signatures of Prohead I and Prohead II indicate a common alpha/beta fold for residues 104-385, and a strongly conserved tertiary structure. The Raman difference spectrum between Prohead I and Prohead II demonstrates that the N-terminal residues 2-103 (Delta-domain) form a predominantly alpha-helical fold devoid of beta-strand. The conformation of the Delta-domain in Prohead I thus resembles that of the previously characterized scaffolding proteins of Salmonellaphage P22 and Bacillus phage phi29 and suggests an analogous architectural role in mediating the assembly of a properly dimensioned precursor shell. The Prohead II --> Head II transition is accompanied by significant reordering of both the secondary and tertiary structures of 104-385, wherein a large increase occurs in the percentage of beta-strand (from 38 to 45%), and a marginal increase is observed in the percentage of alpha-helix (from 27 to 31%). Both are at the expense of unordered chain segments. Residue environments affected by HK97 shell maturation include the unique cysteine (Cys 362) and numerous tyrosines and tryptophans. The tertiary structural reorganization is reminiscent of that observed for the procapsid --> capsid transformation of P22. The Raman signatures of aqueous and crystalline Head II reveal no significant differences between the crystal and solution structures.     

Maturation dynamics of a viral capsid: visualization of transitional intermediate states

Typical of DNA bacteriophages and herpesviruses, HK97 assembles in two stages: polymerization and maturation. First, capsid protein polymerizes into closed shells; then, these precursors mature into larger, stabler particles. Maturation is initiated by proteolysis, producing a metastable particle primed for expansion-the major structural transition. We induced expansion in vitro by acidic pH and monitored the resulting changes by time-resolved X-ray diffraction and cryo-electron microscopy. The transition, which is not synchronized over the population, proceeds in a series of stochastically triggered subtransitions. Three distinct intermediates were identified, which are comparable to transitional states in protein folding. The intermediates' structures reveal the molecular events occurring during expansion. Integrated into a movie (see Dynamic Visualization below), they show capsid maturation as a dynamic process.     

Critical Salt Bridges Guide Capsid Assembly,Stability, and Maturation Behavior in Bacteriophage HK97

HK97 is a double-stranded DNA bacteriophage that undergoes dramatic conformational changes during viral capsid maturation and for which x-ray structures, at near atomic resolution, of multiple intermediate and mature capsid states are available. Both amide H/²H exchange and crystallographic comparisons between the pre-expanded Prohead II particles and the expanded Head II of bacteriophage HK97 revealed quaternary interactions that remain fixed throughout maturation and appear to maintain intercapsomer integrity at all quasi- and icosahedral 3-fold axes. These 3-fold staples are formed from Arg and Glu residues and a metal binding site. Mutations of either Arg-347 or Arg-194 or a double mutation of E344Q and E363A resulted in purification of the phage in capsomer form (hexamers and pentamers). Mutants that did assemble had both decreased thermal stability and decreased in vitro expansion rates. Amide H/²H exchange mass spectrometry showed that in the wild type capsid some subunits had a bent “spine” helix (highly exchanging), whereas others were straight (less exchanging). Similar analysis of the never assembled mutant capsomers showed uniform amide exchange in all of these that was higher than that of the straight spine helices (characterized in more mature intermediates), suggesting that the spine helix is somewhat bent prior to capsid assembly. The result further supports a previously proposed mechanism for capsid expansion in which the delta domains of each subunit induce a high energy intermediate conformation, which now appears to include a bent helix during capsomer assembly.The viral capsid, particularly in double-stranded DNA bacteriophage, requires a highly stable macromolecular structure capable of encapsulating genome at near liquid crystalline density. Viral capsids are composed of hundreds to thousands of individual subunits that efficiently assemble into a closed capsid form often of a highly symmetrized icosahedral geometry, avoiding kinetic traps that would result in increased off-pathway assemblies. Recent studies have proposed that capsid assembly is mediated by weak intersubunit interactions that nucleate larger assembly intermediates, resulting in a considerably more stable capsid form due to a favorable geometry with a more constrained network of interactions. Measurements in systems such as cowpea chlorotic mottle virus, hepatitis B virus, and the bacteriophages P22 and HK97 have estimated the association energy of the initial assembly interaction between two subunits at 2–5 kcal/mol, which is seemingly low for a robust assembly product (1–5). An entropically driven process based on burial of hydrophobic surfaces was considered the driving force for the initial weak interactions with subsequent nucleation and elongation reactions leading to assembly of the full capsid (2, 6). Most complex viruses undergo a staged assembly process involving conformational transitions that occur after the initial assembly of a procapsid (7). The process is known as virion maturation. The interplay between interactions necessary for the initial assembly of capsomers into the procapsid and those that facilitate capsid maturation have been poorly understood, but recent crystal structures of procapsid and mature capsid states of HK97 allowed us to evaluate the structural properties that may facilitate maturation.HK97 is an amenable system for the study of capsid assembly and maturation. Symmetric procapsid particles can be assembled in Escherichia coli with the expression of just two gene products, gp4 (protease) and gp5 (capsid subunit). Maturation can then be followed in vitro by lowering the pH or chemically perturbing the procapsids. Unlike bacteriophages such as P22 that assemble their capsids directly from individual monomeric subunits, HK97 subunits initially assemble into capsomers composed of six-subunit (hexamers) or five-subunit (pentamers) oligomers. Twelve pentamers and 60 hexamers then assemble to form an icosahedral capsid with a triangulation number of 7 laevo, although a portal complex substitutes one of the pentamers during in vivo assembly. Residues 2–103 at the N terminus of the subunit, referred to as the delta domain, is thought to serve the same role as the scaffolding proteins identified for other phage in the assembly process (8). Capsomers then assemble, packaging the protease (gp4), to form the initial procapsid, Prohead I (P-I).¹ If the expression is done without the protease or with an inactive (by mutation) protease, this step is reversible (Fig. 1). The equilibrium of this assembly can be controlled in vitro with specific buffers and concentrations that favor either the capsomer or the capsid form (9). Expression with an active protease leads to proteolysis of the delta domains in the assembled P-I state followed by autodigestion of the protease and diffusion of the fragments from the particle. P-I then undergoes subtle structural adjustments, resulting in the Prohead II state composed entirely of the cleaved gp5* subunits (10, 11). At this stage of assembly in vivo, concatameric double-stranded DNA is packaged through a portal complex (composed of gp3 subunits) that fits into a single 5-fold vertex of the capsid. We used an HK97 construct that lacks gp3, so the purified Prohead II capsid is icosahedrally symmetric and cannot package DNA. Purified P-II can be matured in vitro using low pH and other chemical perturbation methods. During maturation, conformational changes in the subunits and their interactions result in large scale expansion and morphological changes in the capsid. The diameter of the capsid shell increases from 540 Å in P-II to 660 Å in Head II (H-II), the fully expanded particle form (12, 13). Intermediate particle forms can be trapped during the expansion and were previously characterized with a variety of biophysical techniques including cryo-EM microscopy (14, 15), x-ray crystallography (12, 13, 16), and small angle x-ray scattering (16–18). During the expansion process, self-catalyzed covalent cross-links are formed through isopeptide bond formation between Lys-169 and Asn-356 of different subunits situated on adjacent capsomers (19). The reaction is promoted by Glu-363, which is adjacent to the bonding residues and functions as a proton acceptor. Cross-linking during maturation was previously shown by differential scanning calorimetry (DSC) to greatly enhance the thermal stability of HK97 (5). In addition to covalent bonding, the H-II has significantly more buried surface area than P-II as seen in the highly intercalated intersubunit interactions depicted in the previous 3.44-Å structure of Head II (13, 20). A cross-link-defective mutant, K169Y, stills undergoes particle expansion, reaching the penultimate particle form, termed Head I (H-I), which has nearly identical conformations of hexamer capsomers but less extruded pentamers than H-II (16). H-I was used for all H/²H exchange studies instead of H-II because the cross-links in H-II dramatically affect the efficiency of proteolysis required for the mass spectrometry-based experiment (12, 20).Open in a separate windowFig. 1.HK97 assembly and expansion pathway. The schematic diagram depicts the assembly and expansion of HK97 in an E. coli expression system lacking the portal protein and other machinery required for genome packaging. 42-kDa subunits assemble into hexamer and pentamer capsomers, which then assemble into an initial icosahedral procapsid shell, P-I. Proteolytic cleavage of the delta domain of each subunit results in the formation of the metastable intermediate form P-II, which is able to undergo in vitro maturation when perturbed by various chemical agents. WT expansion proceeds through EI, balloon, and ultimately H-II forms, an expansion process that involves covalent cross-linking. K169Y mutant P-II proceeds through EI to the H-I form without any cross-linking occurring. Other than the lack of cross-links, H-I is identical to balloon.It was hypothesized that for highly intercalated mature capsid forms such as that seen in bacteriophage HK97 early procapsid intermediates are necessary for initial positioning of subunits before conformational changes can facilitate a protein architecture with increased stability. We recently showed with amide H/²H exchange and crystallographic comparisons between the pre-expanded P-II particles and the mature H-II that maturation is probably guided by tertiary structure twisting and secondary structure changes around a fixed set of intercapsomer interactions that surround all quasi- and icosahedral 3-fold axes in the capsid shell (12). The major interactions that appear to facilitate these “3-fold staples” include two sets of salt bridges and a putative metal binding site (Fig. 2). The salt bridge interactions are between residues Glu-344 and Arg-194 and between residues Glu-363 and Arg-347. Glutamate 363 serves dual roles as it is involved in both a salt bridge with Arg-347 and serves as a proton acceptor that catalyzes the isopeptide bond formation (21). The metal binding site is formed by 3-fold related glutamates at position 348 interacting with a sphere of electron density at high σ level in the P-II crystal structure (12). Although comparable density for metal ions is not present at the equivalent position in crystal structures of the late intermediates, the positions of the glutamates are nearly identical, indicating a stable interaction with some mechanism for neutralizing the negative charge repulsion. In contrast to the near identical conformations of the residues at the 3-fold interface, the rest of the subunit was shown to undergo a large scale twisting motion, causing hinging in all three P-domain β-strands (see Fig. 8A for domain nomenclature). These data imply that interactions at the 3-fold interface may be crucial in assembling the capsid from individual capsomers as well as providing a fixed point from which subunits bend while maintaining intercapsomer contacts.Open in a separate windowFig. 2.Importance of 3-fold intercapsomer contacts. A, P-II capsid from previously solved 3.65-Å crystal structure rendered in low resolution in chimera. Two hexon subunits (subunits a and f, yellow and green, respectively) and one penton subunit (orange) that form a quasi-3-fold interaction are shown as ribbons. B, zoomed in view of quasi-3-fold interaction between the two hexamer subunits and one pentamer subunit as highlighted in A. The view is from inside the capsid, 180° rotated from the view shown in A. Residues involved in salt bridges as well as a putative metal binding site (Glu-348) are labeled accordingly. C, table identifying various mutations made to perturb 3-fold contacts. The phenotypes following protein expression are identified. Mutants are distinguished as to whether they were purified as capsids or capsomers (hexamers and pentamers) following protein expression. Data for the Glu-363 mutants are from Dierkes et al. (21).Open in a separate windowFig. 8.Solvent accessibility of R347N capsomer spine helix. A, subunit C of Prohead II is shown with the major domains labeled. Residues 206–216 of the spine helix are colored orange. B, mass envelopes for P-II and H-I particle forms as well as the R347N capsomers following 5 min of exchange. The top spectrum is non-deuterated P-II. C, H/²H exchange results of the residues colored orange are plotted for the R347N capsomers (orange curve) and compared with the solvent accessibility curves for the same fragment in the P-II capsid state, EI, and the nearly mature H-I capsid form. D, the solvent accessibility of the same spine helix fragment is shown for both the R347N capsomers and WT capsomers that were disassembled from the P-I state.Here we confirmed this role for the 3-fold interactions by mutagenesis of relevant residues and characterized the resulting assembly products, thermal stabilities, and maturation kinetics. Some of the mutants did not assemble into particles following the formation of capsomers (e.g. R347N). Capsomers were then purified, and the amide exchange of the spine helices was analyzed with H/²H exchange coupled to mass spectrometry (22–24). Previous data illustrated a direct correlation between increased H/²H exchange and an increased bend in the helix conformation (12, 20). Amide exchange of the spine helix in the mutant capsomers was compared with previously characterized particle forms as well as P-I and WT capsomers disassembled from P-I.     

Molecular rearrangements involved in the capsid shell maturation of bacteriophage T7

Maturation of dsDNA bacteriophages involves assembling the virus prohead from a limited set of structural components followed by rearrangements required for the stability that is necessary for infecting a host under challenging environmental conditions. Here, we determine the mature capsid structure of T7 at 1 nm resolution by cryo-electron microscopy and compare it with the prohead to reveal the molecular basis of T7 shell maturation. The mature capsid presents an expanded and thinner shell, with a drastic rearrangement of the major protein monomers that increases in their interacting surfaces, in turn resulting in a new bonding lattice. The rearrangements include tilting, in-plane rotation, and radial expansion of the subunits, as well as a relative bending of the A- and P-domains of each subunit. The unique features of this shell transformation, which does not employ the accessory proteins, inserted domains, or molecular interactions observed in other phages, suggest a simple capsid assembling strategy that may have appeared early in the evolution of these viruses.     

A conformational switch involved in maturation of Staphylococcus aureus bacteriophage 80α capsids

Bacteriophages are involved in many aspects of the spread and establishment of virulence factors in Staphylococcus aureus, including the mobilization of genetic elements known as S. aureus pathogenicity islands (SaPIs), which carry genes for superantigen toxins and other virulence factors. SaPIs are packaged into phage-like transducing particles using proteins supplied by the helper phage. We have used cryo-electron microscopy and icosahedral reconstruction to determine the structures of the procapsid and the mature capsid of 80α, a bacteriophage that can mobilize several different SaPIs. The 80α capsid has T = 7 icosahedral symmetry with the capsid protein organized into pentameric and hexameric clusters that interact via prominent trimeric densities. The 80α capsid protein was modeled based on the capsid protein fold of bacteriophage HK97 and fitted into the 80α reconstructions. The models show that the trivalent interactions are mediated primarily by a 22-residue β hairpin structure called the P loop that is not found in HK97. Capsid expansion is associated with a conformational switch in the spine helix that is propagated throughout the subunit, unlike the domain rotation mechanism in phage HK97 or P22.     

Coat protein fold and maturation transition of bacteriophage P22 seen at subnanometer resolutions

Bacteriophage P22 is a prototypical biological machine used for studying protein complex assembly and capsid maturation. Using cryo-EM, we solved the structures of P22 before and after the capsid maturation at 8.5 A and 9.5 A resolutions, respectively. These structures allowed visualization of alpha-helices and beta-sheets from which the capsid protein fold is derived. The capsid fold is similar to that of the coat protein of HK97 bacteriophage. The cryo-EM shows that a large conformational change of the P22 capsid during maturation transition involves not only the domain movement of individual subunits, but also refolding of the capsid protein.     

Structure and assembly of the capsid of bacteriophage P22.

Identification of the genes and proteins involved in phage P22 formation has permitted a detailed analysis of particle assembly, revealing some unexpected aspects. The polymerization of the major coat protein (gene 5 product) into an organized capsid is directed by a scaffolding protein (gene 8 product) which is absent from mature phage. The resulting capsid structure (prohead) is the precursor for DNA encapsidation. All of the scaffolding protein exits from the prohead in association with DNA packaging. These molecules then recycle, directing further rounds of prohead assembly. The structure of the prohead has been studied by electron microscopy of thin sections of phage infected cells, and by low angle X-ray scattering of concentrated particles. The results show that the prohead is a double shell structure, or a ball within a shell. The inner ball or shell is composed of the scaffolding protein while the outer shell is composed of coat protein. The conversion from prohead to mature capsid is associated with an expansion of the coat protein shell. It is possible that the scaffolding protein molecules exit through the capsid lattice. When DNA encapsidation within infected cells is blocked by mutation, scaffolding protein is trapped in proheads and cannot recycle. Under these conditions, the rate of synthesis of gp8 increases, so that normal proheads continue to form. These results suggest that free scaffolding protein negatively regulates its own further synthesis, providing a coupling between protein synthesis and protein assembly.     

Bacteriophage T5 structure reveals similarities with HK97 and T4 suggesting evolutionary relationships

Evolutionary relationships between viruses may be obscure by protein sequence but unmasked by structure. Analysis of bacteriophage T5 by cryo-electron microscopy and protein sequence analysis reveals analogies with HK97 and T4 that suggest a mosaic of such connections. The T5 capsid is consistent with the HK97 capsid protein fold but has a different geometry, incorporating three additional hexamers on each icosahedral facet. Similarly to HK97, the T5 major capsid protein has an N-terminal extension, or Delta-domain that is missing in the mature capsid, and by analogy with HK97, may function as an assembly or scaffold domain. This Delta-domain is predicted to be largely coiled-coil, as for that of HK97, but is approximately 70% longer correlating with the larger capsid. Thus, capsid architecture appears likely to be specified by the Delta-domain. Unlike HK97, the T5 capsid binds a decoration protein in the center of each hexamer similarly to the "hoc" protein of phage T4, suggesting a common role for these molecules. The tail-tube has unusual trimeric symmetry that may aid in the unique two-stage DNA-ejection process, and joins the tail-tip at a disk where tail fibers attach. This intriguing mix of characteristics embodied by phage T5 offers insights into virus assembly, subunit function, and the evolutionary connections between related viruses.     

Conservation of the capsid structure in tailed dsDNA bacteriophages: the pseudoatomic structure of phi29

Bacteriophage phi29 is one of the smallest and simplest known dsDNA phages, making it amenable to structural investigations. The three-dimensional structure of a fiberless, isometric variant has been determined to 7.9 A resolution by cryo-electron microscopy (cryo-EM), allowing the identification of alpha helices and beta sheets. Their arrangement indicates that the folds of the phi29 and bacteriophage HK97 capsid proteins are similar except for an additional immunoglobulin-like domain of the phi29 protein. An atomic model that incorporates these two domains fits well into the cryo-EM density of the T = 3, fiberless isometric phi29 particle, and cryo-EM structures of fibered isometric and fiberless prolate prohead phi29 particles at resolutions of 8.7 A and 12.7 A, respectively. Thus, phi29 joins the growing number of phages that utilize the HK97 capsid structure, suggesting that this protein fold may be as prevalent in capsids of dsDNA phages as the jelly roll fold is in eukaryotic viruses.     

An elastic network model of HK97 capsid maturation

The structure of the capsid of bacteriophage HK97 has been solved at various stages of maturity by crystallography and cryo-electron microscopy, and has been reported previously in the literature. Typically the capsid assembles through polymerization and maturation processes. Maturation is composed of proteolytic cleavages to the precursor capsid (called Prohead II), expansion triggered by DNA packaging (in which the largest conformational changes of the capsid appear), and covalent cross-links of neighboring subunits to create the mature capsid called Head II. We apply a coarse-grained elastic network interpolation (ENI) to generate a feasible pathway for conformational change from Prohead II to Head II. The icosahedral symmetry of the capsid structure offers a significant computational advantage because it is not necessary to consider the whole capsid structure but only an asymmetric unit consisting of one hexamer plus an additional subunit from an adjacent pentamer. We also analyze normal modes of the capsid structure using an elastic network model which is also subject to symmetry constraints. Using our model, we can visualize the smooth evolution of capsid expansion and revisit in more detail several interesting geometric changes recognized in early experimental works such as rigid body motion of two compact domains (A and P) with two refolding extensions (N-arm and E-loop) and track the approach of the two particular residues associated with isopeptide bonds that make hexagonal cross-links in Head II. The feasibility of the predicted pathway is also supported by the results of our normal mode analysis.     

Bacteriophage HK97 structure: wholesale covalent cross-linking between the major head shell subunits.

We describe initial genetic and structural characterizations of HK97, a temperate bacteriophage of Escherichia coli. We isolated 28 amber mutants, characterized them with respect to what phage-related structures they make, and mapped many of them to restriction fragments of genomic DNA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of HK97 virions revealed nine different protein species plus a substantial amount of material that failed to enter the gel, apparently because it is too large. Five proteins are tail components and are assigned functions as tail fiber subunit, tail length template, and major shaft subunit (two and possibly three species). The four remaining proteins and the material that did not enter the gel are head components. One of these proteins is assigned as the portal subunit, and the remaining three head proteins in the gel and the material that did not enter the gel are components of the head shell. All of the head shell protein species have apparent molecular masses well in excess of 100 kDa; they share amino acid sequence with each other and also with a 42-kDa protein that is found in infected lysates and as the major component of prohead structures that accumulate in infections by one of the amber mutants. We propose that all of the head shell species found in mature heads are covalently cross-linked oligomers derived from the 42-kDa precursor during head shell maturation.