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.     

Control of virus assembly: HK97 "Whiffleball" mutant capsids without pentons

The capsid of Escherichia coli bacteriophage HK97 assembles as a 420 subunit icosahedral shell called Prohead I which undergoes a series of maturation steps, including proteolytic cleavage, conformational rearrangements, and covalent cross-linking among all the subunits to yield the highly stable mature Head II shell. Prohead I have been shown to assemble from pre-formed hexamers and pentamers of the capsid protein subunit. We report here the properties of a mutant of the capsid protein, E219K, which illuminate the assembly of Prohead I. The mutant capsid protein is capable of going through all of the biochemically and morphologically defined steps of capsid maturation, and when it is expressed by itself from a plasmid it assembles efficiently into a Prohead I that is morphologically indistinguishable from the wild-type Prohead I, with a full complement of both hexamers and pentamers. Unlike the wild-type Prohead I, when the mutant structure is dissociated into capsomers in vitro, only hexamers are found. When such preparations are put under assembly conditions, these mutant hexamers assemble into "Whiffleballs", particles that are identical with Prohead I except that they are missing the 12 pentamers. These Whiffleballs can even be converted to Prohead I by specifically binding wild-type pentamers. We argue that the ability of the mutant hexamers to assemble in the absence of pentamers implies that they retain a memory of their earlier assembled state, most likely as a conformational difference relative to assembly-naive hexamers. The data therefore favor a model in which Prohead I assembly is regulated by conformational switching of the hexamer.     

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.     

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.     

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.     

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.     

Mutational Analysis of a Conserved Glutamic Acid Required for Self-Catalyzed Cross-Linking of Bacteriophage HK97 Capsids

The capsid of bacteriophage HK97 is stabilized by ∼400 covalent cross-links between subunits which form without any action by external enzymes or cofactors. Cross-linking only occurs in fully assembled particles after large-scale structural changes bring together side chains from three subunits at each cross-linking site. Isopeptide cross-links form between asparagine and lysine side chains on two subunits. The carboxylate of glutamic acid 363 (E363) from a third subunit is found ∼2.4 Å from the isopeptide bond in the partly hydrophobic pocket that contains the cross-link. It was previously reported without supporting data that changing E363 to alanine abolishes cross-linking, suggesting that E363 plays a role in cross-linking. This alanine mutant and six additional substitutions for E363 were fully characterized and the proheads produced by the mutants were tested for their ability to cross-link under a variety of conditions. Aspartic acid and histidine substitutions supported cross-linking to a significant extent, while alanine, asparagine, glutamine, and tyrosine did not, suggesting that residue 363 acts as a proton acceptor during cross-linking. These results support a chemical mechanism, not yet fully tested, that incorporates this suggestion, as well as features of the structure at the cross-link site. The chemically identical isopeptide bonds recently documented in bacterial pili have a strikingly similar chemical geometry at their cross-linking sites, suggesting a common chemical mechanism with the phage protein, but the completely different structures and folds of the two proteins argues that the phage capsid and bacterial pilus proteins have achieved shared cross-linking chemistry by convergent evolution.A common feature of the assembly pathways of all tailed double-stranded DNA bacteriophages and herpesviruses (45) is that they package their chromosomes into unstable precursors called procapsids or proheads, which mature into virions. The virion DNA is packed to a very high density (∼0.5 g/ml) (13), so the completed DNA-filled capsids must be specifically reinforced or stabilized during maturation to withstand the very high effective internal “DNA pressure” (20, 41, 49). Virions also must resist the chemical and physical stresses of the environment outside their hosts. Phage capsids achieve this stabilization by several different mechanisms, all of which include large conformational changes in the capsid. Some phages, like P22 (14) and T7 (4), achieve stabilization by conformational changes alone. For other phages, such as T4 (43) and λ (42), full stabilization requires the binding of so-called “decoration” proteins to the outside of the capsid (44). Capsid stabilization may also occur by forming covalent cross-links between capsid protein subunits, as in phage HK97 (10, 11, 36, 51). The HK97 cross-links are isopeptide bonds between asparagine and lysine side chains on neighboring subunits that tie together structural elements on the inside of the shell to elements on the outside, thus generating a chain-mail-like network of connections among capsid subunits and making a very stable capsid (39, 51). This type of amide bond was thought to occur only in phage capsid proteins, where they occur frequently (23), but it has also been identified recently within the subunits of the pili of Streptococcus pyogenes and may be widespread in related pilus proteins (26).HK97 is a λ-like phage of Escherichia coli (7, 25) with a long noncontractile tail and a T=7 icosahedral capsid (5). HK97 procapsids assemble from the major capsid protein, the capsid protease, and the portal protein as outlined in Fig. ​Fig.1.1. Assembly occurs during phage infection or when the same proteins are expressed from plasmids in E. coli. After assembly of Prohead I, the protease cleaves interior domains from the major capsid protein and digests itself, yielding the procapsid, Prohead II, which contains only cleaved major capsid protein. Prohead II is the natural substrate for DNA packaging during phage infection, and it matures during packaging by a process called expansion and transforms into Head II, for which the X-ray structure has been determined (22, 51). Expansion can also be induced chemically in vitro and has been studied extensively, both biochemically and in multiple cryo-electron microscopy (cryo-EM) and X-ray structures (5, 6, 15, 16, 31-33, 39). Expansion involves extensive rearrangements of the capsid subunits which change the round, thick-walled ∼550-Å Prohead II into the angular, thin-walled ∼650Å Head II (Fig. ​(Fig.1).1). Capsid cross-links are made during expansion.Open in a separate windowFIG. 1.Assembly and maturation of the HK97 capsid. Three proteins are required to assemble Prohead II, the HK97 procapsid. These are the major capsid protein (gp5, 42 kDa), the protease (gp4, 25 kDa), and the portal or connector protein (gp3, 46 kDa) (12). The major capsid protein folds and assembles into hexamers and pentamers (capsomers collectively), which coassemble with the protease to form Prohead I. The protease removes 102 residues of each capsid subunit and destroys itself to create Prohead II. The portal complex normally occupies the position of one pentamer and is required for viable phage assembly, but the portal protein is optional for making particles from plasmid-expressed genes (5, 11). Prohead II expands to form the mature capsid, Head II, normally during DNA packaging, but also in vitro as described in Results.The high-resolution structure of the HK97 mature capsid provides a structural basis for understanding many aspects of assembly and cross-linking (22, 51). The subunits of the mature capsid have a continuous core with a dominant long alpha-helix and two extended arms, the N-arm and the long β-hairpin or E-loop (for Extended loop), as shown in Fig. ​Fig.2A.2A. Cross-links cannot form until a complete particle is assembled—this can be easily understood by looking at the Head II subunit by itself and the arrangement of subunits in the mature particle (Fig. 2A and B). The three residues required for cross-linking are far apart in an isolated subunit, as they are in an individual hexamer or pentamer of capsid protein from which the proheads assemble. At least three capsomers (hexamers or pentamers of capsid protein) must assemble in a manner like that shown in Fig. 2B and C in order to bring together the three residues that will participate in a given cross-link. This is not achieved directly; the capsomers initially assemble into proheads, in which the two residues that will cross-link are still separated by more than 30 Å (6). Cross-linking does not occur until the conformational rearrangements of the expansion reaction bring asparagine 356 (N356) and lysine 169 (K169) close to glutamic acid 363 (E363) in an active site-like cavity. We argue here that E363 has a catalytic role in the cross-linking reaction.Open in a separate windowFIG. 2.Three-dimensional models of the sites of capsid cross-linking in HK97. Views of HK97 cross-link sites based on the refined structure (PDB ID 1OHG) (22) show the proximity of the side chain of glutamate E363 to the joined residues. (A) An HK97 Head II subunit (D chain) in isolation shows the three side chains involved in capsid cross-linking: an asparagine (N356, dark blue) at the end of the P-domain, a lysine (K169, red) near the tip of the E-loop, and a glutamate (E363, orange) protruding from a β-sheet (β-J) in the P-domain. HK97 cross-links occur at sites located around the threefold and quasi-threefold axes of symmetry of the HK97 capsid, and one such trio of cross-links is shown in panels B and C. (B) Cross-links formed around a threefold axis (red-filled triangle) and their context among the three hexons that comprise a flat icosahedral face of the particle. The hexamer chains are labeled A to F, and the locations of several quasi-threefold axes are marked with open triangles. Views are from the inside of a capsid, looking outward. (C) Close-up view of the area boxed in panel B. The chains joined by the highlighted cross-links are colored identically in pairs: a lysine from a D chain is joined to an asparagine in an E chain from a neighboring hexamer, aided by a glutamate in the D chain of a neighboring hexamer. Side chain oxygens in the glutamates and cross-links are indicated in red. In the lower of the three cross-link sites, the side chains of three hydrophobic residues that surround the cross-links are shown in space-filling representations; these are residues V163, L361, and M339 and are labeled in red. Images made using SPDBV 3.7 (21) and the Persistence of Vision Raytracer (POV-Ray Mac 3.6 [http://www.povray.org/]).In the Head II X-ray model (22), one of the cross-link residues, N356, is near the outer end of the P-domain, sandwiched within the subunit interface near junctions where three hexamers (or two hexamers and a pentamer) meet (see Fig. ​Fig.2),2), while the other, K169, projects into the subunit interface from near the tip of the E-loop. The third residue involved in cross-linking, E363, is located on a third subunit, which lies across the local capsomer-capsomer interface from N356 (Fig. 2B and C) and one carboxylate oxygen of E363 is found only 2.5 Å away from the isopeptide bond. This arrangement is found in each of the seven unique (quasi-equivalent) cross-link sites in the Head II model (22, 51). Changing E363 to alanine (E363A) was found to abolish cross-linking (51), suggesting that E363 may have an important chemical role in the cross-linking reaction. Further support for a role of E363 in cross-linking comes from evidence that it appears to be conserved and easily identified in alignments of the HK97 capsid protein sequence with other capsid proteins in GenBank. For example, a BLAST search of the nonredundant protein database with the mature HK97 capsid protein sequence yields ∼60 non-self-matches, and 43 of these potential homologs appear to possess all three cross-linking residues (matching K169, N356, and E363 in HK97), notably including several bacteriophages for which there is experimental evidence of capsid cross-linking: D3 (19), XP10 (54), BFK20 (3), and Φ11b (2). The findings we present here suggest that both the size and the character of residue 363 can influence assembly and maturation of HK97 capsids, but only glutamate is capable of promoting efficient cross-linking. In the analogous Asn-Lys isopeptide bonds recently identified in the pili of gram-positive bacteria, a conserved glutamic (or aspartic) acid residue is also poised nearby and implicated in cross-linking, suggesting a common mechanism.     

Diversity and identity of mechanical properties of icosahedral viral capsids studied with elastic network normal mode analysis

We analyze the mechanical properties and putative dynamical fluctuations of a variety of viral capsids comprising different sizes and quasi-equivalent symmetries by performing normal mode analysis using the elastic network model. The expansion of the capsid to a swollen state is studied using normal modes and is compared with the experimentally observed conformational change for three of the viruses for which experimental data exist. We show that a combination of one or two normal modes captures remarkably well the overall translation that dominates the motion between the two conformational states, and reproduces the overall conformational change. We observe for all of the viral capsids that the nature of the modes is different. In particular for the T=7 virus, HK97, for which the shape of the capsid changes from spherical to faceted polyhedra, two modes are necessary to accomplish the conformational transition. In addition, we extend our study to viral capsids with other T numbers, and discuss the similarities and differences in the features of virus capsid conformational dynamics. We note that the pentamers generally have higher flexibility and propensity to move freely from the other capsomers, which facilitates the shape adaptation that may be important in the viral life cycle.     

A thermally induced phase transition in a viral capsid transforms the hexamers, leaving the pentamers unchanged

Scanning calorimetry combined with cryo-electron microscopy affords a powerful approach to investigating hierarchical interactions in multi-protein complexes. Calorimetry can detect the temperatures at which certain interactions are disrupted and cryo-EM can reveal the accompanying structural changes. The procapsid of bacteriophage HK97 (Prohead I) is a 450A-diameter shell composed of 60 hexamers and 12 pentamers of gp5, organized with icosahedral symmetry. Gp5 consists of the N-terminal Delta-domain (11kDa) and gp5* (31 kDa): gp5* forms the contiguous shell from which clusters of Delta-domains extend inwards. At neutral pH, Prohead I exhibits an endothermic transition at 53 degrees C with an enthalpy change of 14 kcal/mole (of gp5 monomer). We show that this transition is reversible. To capture its structural expression, we incubated Prohead I at 60 degrees C followed by rapid freezing and, by cryo-EM, observed a capsid species 10% larger than Prohead I. At 11A resolution, visible changes are confined to the gp5 hexamers. Their Delta-domain clusters have disappeared and are presumably disordered, either by unfolding or dispersal. The gp5* hexamer rings are thinned and flattened as they assume the conformation observed in Expansion Intermediate I, a transition state of the normal, proteolysis-induced, maturation pathway. We infer that, at ambient temperatures, the hexamer Delta-domains restrain their gp5* rings from switching to a lower free energy, EI-I-like, state; above 53 degrees, this restraint is overcome. Pentamers, on the other hand, are more stably anchored and resist this thermal perturbation.     

A model-structure of a periplasm-facing state of the NhaA antiporter suggests the molecular underpinnings of pH-induced conformational changes

The Escherichia coli NhaA antiporter couples the transport of H(+) and Na(+) (or Li(+)) ions to maintain the proper pH range and Na(+) concentration in cells. A crystal structure of NhaA, solved at pH 4, comprises 12 transmembrane helices (TMs), arranged in two domains, with a large cytoplasm-facing funnel and a smaller periplasm-facing funnel. NhaA undergoes conformational changes, e.g. after pH elevation to alkaline ranges, and we used two computational approaches to explore them. On the basis of pseudo-symmetric features of the crystal structure, we predicted the structural architecture of an alternate, periplasm-facing state. In contrast to the crystal structure, the model presents a closed cytoplasmic funnel, and a periplasmic funnel of greater volume. To examine the transporter functional direction of motion, we conducted elastic network analysis of the crystal structure and detected two main normal modes of motion. Notably, both analyses predicted similar trends of conformational changes, consisting of an overall rotational motion of the two domains around a putative symmetry axis at the funnel centers, perpendicular to the membrane plane. This motion, along with conformational changes within specific helices, resulted in closure at the cytoplasmic end and opening at the periplasmic end. Cross-linking experiments, performed between segments on opposite sides of the cytoplasmic funnel, revealed pH-dependent interactions consistent with the proposed conformational changes. We suggest that the model-structure and predicted motion represent alkaline pH-induced conformational changes, mediated by a cluster of evolutionarily conserved, titratable residues, at the cytoplasmic ends of TMs II, V, and IX.     

Rigid-cluster models of conformational transitions in macromolecular machines and assemblies

We present a rigid-body-based technique (called rigid-cluster elastic network interpolation) to generate feasible transition pathways between two distinct conformations of a macromolecular assembly. Many biological molecules and assemblies consist of domains which act more or less as rigid bodies during large conformational changes. These collective motions are thought to be strongly related with the functions of a system. This fact encourages us to simply model a macromolecule or assembly as a set of rigid bodies which are interconnected with distance constraints. In previous articles, we developed coarse-grained elastic network interpolation (ENI) in which, for example, only Calpha atoms are selected as representatives in each residue of a protein. We interpolate distance differences of two conformations in ENI by using a simple quadratic cost function, and the feasible conformations are generated without steric conflicts. Rigid-cluster interpolation is an extension of the ENI method with rigid-clusters replacing point masses. Now the intermediate conformations in an anharmonic pathway can be determined by the translational and rotational displacements of large clusters in such a way that distance constraints are observed. We present the derivation of the rigid-cluster model and apply it to a variety of macromolecular assemblies. Rigid-cluster ENI is then modified for a hybrid model represented by a mixture of rigid clusters and point masses. Simulation results show that both rigid-cluster and hybrid ENI methods generate sterically feasible pathways of large systems in a very short time. For example, the HK97 virus capsid is an icosahedral symmetric assembly composed of 60 identical asymmetric units. Its original Hessian matrix size for a Calpha coarse-grained model is >(300,000)(2). However, it reduces to (84)(2) when we apply the rigid-cluster model with icosahedral symmetry constraints. The computational cost of the interpolation no longer scales heavily with the size of structures; instead, it depends strongly on the minimal number of rigid clusters into which the system can be decomposed.     

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.     

Opening and closing of a toroidal group II chaperonin revealed by a symmetry constrained elastic network model

Recently, the atomic structures of both the closed and open forms of Group 2 chaperonin protein Mm‐cpn were revealed through crystallography and cryo‐electron microscopy. This toroidal‐like chaperonin is composed of two eightfold rings that face back‐to‐back. To gain a computational advantage, we used a symmetry constrained elastic network model (SCENM), which requires only a repeated subunit structure and its symmetric connectivity to neighboring subunits to simulate the entire system. In the case of chaperonin, only six subunits (i.e., three from each ring) were used out of the eight subunits comprising each ring. A smooth and symmetric pathway between the open and closed conformations was generated by elastic network interpolation (ENI). To support this result, we also performed a symmetry‐constrained normal mode analysis (NMA), which revealed the intrinsic vibration features of the given structures. The NMA and ENI results for the representative single subunit were duplicated according to the symmetry pattern to reconstruct the entire assembly. To test the feasibility of the symmetry model, its results were also compared with those obtained from the full model. This study allowed the folding mechanism of chaperonin Mm‐cpn to be elucidated by SCENM in a timely manner.     

Influence of nonuniform geometry on nanoindentation of viral capsids

A series of recent nanoindentation experiments on the protein shells (capsids) of viruses has established atomic force microscopy (AFM) as a useful framework for probing the mechanics of large protein assemblies. Specifically these experiments provide an opportunity to study the coupling of the global assembly response to local conformational changes. AFM experiments on cowpea chlorotic mottle virus, known to undergo a pH-controlled swelling conformational change, have revealed a pH-dependent mechanical response. Previous theoretical studies have shown that homogeneous changes in shell geometry can play a significant role in the mechanical response. This article develops a method for accurately capturing the heterogeneous geometry of a viral capsid and explores its effect on mechanical response with a nonlinear continuum elasticity model. Models of both native and swollen cowpea chlorotic mottle virus capsids are generated from x-ray crystal structures, and are used in finite element simulations of AFM indentation along two-, three-, and fivefold icosahedral symmetry orientations. The force response of the swollen capsid model is observed to be softer by roughly a factor of two, significantly more nonlinear, and more orientation-dependent than that of a native capsid with equivalent elastic moduli, demonstrating that capsid geometric heterogeneity can have significant effects on the global structural response.     

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.     

Conformational changes in adeno-associated virus type 1 induced by genome packaging

Adeno-associated virus (AAV) is frequently used as a vector for gene therapy. The viral capsid consists of three structural proteins (VP1, VP2, and VP3) that have a common C-terminal core (VP3), with N-terminal extensions of increasing length in VP2 and VP1. The capsid encloses a single-stranded genome of up to 4.7 kb, which is packaged into empty capsids. The N-terminal extension of VP1 carries a phospholipase domain that becomes accessible during infection in the endosomal pathway. We have used cryo-electron microscopy and image reconstruction to determine subnanometer-resolution structures of recombinant AAV1 that has packaged different amounts of a 3. 6-kb recombinant genome. The maps show that the AAV1 capsid undergoes continuous conformational changes upon packaging of the genome. The rearrangements occur at the inner capsid surface and lead to constrictions of the pores at the 5-fold symmetry axes and to subtle movements of the β-sheet regions of the capsid proteins. In fully packaged particles, the genome forms stem-like features that contact the inner capsid surface at the 3-fold symmetry axes. We think that the reorganization of the inner surface has an impact on the viral life cycle during infection, preparing the externalization of phospholipase domains through the pores at the 5-fold symmetry axes and possibly genome release.     

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.     

The mechanism and pathway of pH induced swelling in cowpea chlorotic mottle virus

Normal mode analysis based on a simplified energy function was used to study the swelling process of the icosahedral virus, cowpea chlorotic mottle virus (CCMV). Native state virus particles (coat proteins) of this T=3 icosahedral virus have been shown to undergo a large conformational change to a swollen state when metal ions are removed or the pH is raised. A normal mode analysis based on the native state capsid showed one preferential direction, a breathing mode, that explains the majority of the structural rearrangement necessary to bring the native structure close to the swollen state. From the native form of CCMV, the structure can be displaced along the direction of a single breathing mode by different amounts to create several candidate swollen structures and a putative pathway for virus expansion. The R-factor between these predicted swollen capsid structures and experimental electron density from cryoelectron microscopy (cryo-EM) measurements is then calculated to indicate how well each structure satisfies the experimental measurements on the swollen capsid state. A decrease of the crystallographic R-factor value from approximately 72% to approximately 49% was observed for these simple incremental displacements along the breathing mode. The simultaneous displacement of the native structure along other relevant (symmetric, non-degenerate) modes produce a structure with an R-factor of 45%, which is further reduced to 43.9% after minimization: a value in good accord with models based on the EM data at 28 A resolution. Based on the incrementally expanded structures, a pathway for the swelling process has been proposed. Analysis of the intermediate structures along this pathway indicates a significant loss of interactions at the quasi-3-fold interfaces occurs in the initial stages of the swelling process and this serves as a trigger for the compact to swollen transition. Furthermore, the pH dependent swelling appears to be triggered by the titration of a single residue with an anomalous pK(a) value in the unswollen particle.     

Weak protein-protein interactions are sufficient to drive assembly of hepatitis B virus capsids

Hepatitis B virus (HBV) is an enveloped DNA virus with a spherical capsid (or core). The capsid is constructed from 120 copies of the homodimeric capsid protein arranged with T = 4 icosahedral symmetry. We examined in vitro assembly of purified E. coli expressed HBV capsid protein. After equilibration, concentrations of capsid and dimer were evaluated by size exclusion chromatography. The extent of assembly increased as temperature and ionic strength increased. The concentration dependence of capsid assembly conformed to the equilibrium expression: K(capsid) = [capsid]/[dimer](120). Given the known geometry for HBV capsids and dimers, the per capsid assembly energy was partitioned into energy per subunit-subunit contact. We were able to make three major conclusions. (i) Weak interactions (from -2.9 kcal/mol at 21 degrees C in low salt to -4.4 kcal/mol at 37 degrees C in high salt) at each intersubunit contact result in a globally stable capsid; weak intersubunit interactions may be the basis for the phenomenon of capsid breathing. (ii) HBV assembly is characterized by positive enthalpy and entropy. The reaction is entropy-driven, consistent with the largely hydrophobic contacts found in the crystal structure. (iii) Increasing NaCl concentration increases the magnitude of free energy, enthalpy, and entropy, as if ionic strength were increasing the amount of hydrophobic surface buried by assembly. This last point leads us to suggest that salt acts by inducing a conformational change in the dimer from an assembly-inactive form to an assembly-active form. This model of conformational change linked to assembly is consistent with immunological differences between dimer and capsid.     

Infectious bursal disease virus capsid assembly and maturation by structural rearrangements of a transient molecular switch

Infectious bursal disease virus (IBDV), a double-stranded RNA (dsRNA) virus belonging to the Birnaviridae family, is an economically important avian pathogen. The IBDV capsid is based on a single-shelled T=13 lattice, and the only structural subunits are VP2 trimers. During capsid assembly, VP2 is synthesized as a protein precursor, called pVP2, whose 71-residue C-terminal end is proteolytically processed. The conformational flexibility of pVP2 is due to an amphipathic alpha-helix located at its C-terminal end. VP3, the other IBDV major structural protein that accomplishes numerous roles during the viral cycle, acts as a scaffolding protein required for assembly control. Here we address the molecular mechanism that defines the multimeric state of the capsid protein as hexamers or pentamers. We used a combination of three-dimensional cryo-electron microscopy maps at or close to subnanometer resolution with atomic models. Our studies suggest that the key polypeptide element, the C-terminal amphipathic alpha-helix, which acts as a transient conformational switch, is bound to the flexible VP2 C-terminal end. In addition, capsid protein oligomerization is also controlled by the progressive trimming of its C-terminal domain. The coordination of these molecular events correlates viral capsid assembly with different conformations of the amphipathic alpha-helix in the precursor capsid, as a five-alpha-helix bundle at the pentamers or an open star-like conformation at the hexamers. These results, reminiscent of the assembly pathway of positive single-stranded RNA viruses, such as nodavirus and tetravirus, add new insights into the evolutionary relationships of dsRNA viruses.