Scaffolding protein regulates the polymerization of P22 coat subunits into icosahedral shells in vitro

Coat and scaffolding subunits derived from P22 procapsids have been purified in forms that co-assemble rapidly and efficiently into icosahedral shells in vitro under native conditions. The half-time for this reaction is approximately five minutes at 21 degrees C. The in vitro reaction exhibits the regulated features observed in vivo. Neither coat nor scaffolding subunits alone self-assemble into large structures. Upon mixing the subunits together they polymerize into procapsid-like shells with the in vivo coat and scaffolding protein composition. The subunits in the purified coat protein preparations are monomeric. The scaffolding subunits appear to be monomeric or dimeric. These results confirm that P22 procapsid formation does not proceed through the assembly of a core of scaffolding, which then organizes the coat, but requires copolymerization of coat and scaffolding. To explore the mechanisms of the control of polymerization, shell assembly was examined as a function of the input ratio of scaffolding to coat subunits. The results indicated that scaffolding protein was required for both initiation of shell assembly and continued polymerization. Though procapsids produced in vivo contain about 300 molecules of scaffolding, shells with fewer subunits could be assembled down to a lower limit of about 140 scaffolding subunits per shell. The overall results of these experiments indicate that coat and scaffolding subunits must interact in both the initiation and the growth phases of shell assembly. However, it remains unclear whether during growth the coat and scaffolding subunits form a mixed oligomer prior to adding to the shell or whether this occurs at the growing edge.     

Nucleation and growth phases in the polymerization of coat and scaffolding subunits into icosahedral procapsid shells.

The polymerization of protein subunits into precursor shells empty of DNA is a critical process in the assembly of double-stranded DNA viruses. For the well-characterized icosahedral procapsid of phage P22, coat and scaffolding protein subunits do not assemble separately but, upon mixing, copolymerize into double-shelled procapsids in vitro. The polymerization reaction displays the characteristics of a nucleation limited reaction: a paucity of intermediate assembly states, a critical concentration, and kinetics displaying a lag phase. Partially formed shell intermediates were directly visualized during the growth phase by electron microscopy of the reaction mixture. The morphology of these intermediates suggests that assembly is a highly directed process. The initial rate of this reaction depends on the fifth power of the coat subunit concentration and the second or third power of the scaffolding concentration, suggesting that pentamer of coat protein and dimers or trimers of scaffolding protein, respectively, participate in the rate-limiting step.     

Initiation of P22 procapsid assembly in vivo

The procapsids of all double-stranded DNA phages have a unique portal vertex, which is the locus of DNA packaging and DNA injection. Procapsid assembly is also initiated at this vertex, which is defined by the presence of a cyclic dodecamer of the portal protein. Assembly of the procapsid shell of phage P22 requires the gene 5 coat protein and the gene 8 scaffolding protein. We report here that removal of gene product (gp) 1 portal protein of P22 by mutation does not slow the rate of polymerization of coat and scaffolding subunits into shells, indicating that the portal ring is dispensable for shell initiation. Mutant scaffolding subunits specified by tsU172 copolymerize with coat subunits into procapsids at restrictive temperature, and also correctly autoregulate their synthesis. However, the shell structures formed from the temperature-sensitive scaffolding subunits fail to incorporate the portal ring and the three minor DNA injection proteins. This mutation identifies a domain of the scaffolding protein specifically involved in organization of the portal vertex. The results suggest that it is a complex of the scaffolding protein that initiates procapsid assembly and organizes the portal ring.     

The bacteriophage lambda gpNu3 scaffolding protein is an intrinsically disordered and biologically functional procapsid assembly catalyst

Procapsid assembly is a process whereby hundreds of copies of a major capsid protein assemble into an icosahedral protein shell into which the viral genome is packaged. The essential features of procapsid assembly are conserved in both eukaryotic and prokaryotic complex double-stranded DNA viruses. Typically, a portal protein nucleates the co-polymerization of an internal scaffolding protein and the major capsid protein into an icosahedral capsid shell. The scaffolding proteins are essential to procapsid assembly. Here, we describe the solution-based biophysical and functional characterization of the bacteriophage lambda (λ) scaffolding protein gpNu3. The purified protein possesses significant α-helical structure and appears to be partially disordered. Thermally induced denaturation studies indicate that secondary structures are lost in a cooperative, apparent two-state transition (T_m = 40.6 ± 0.3 °C) and that unfolding is, at least in part, reversible. Analysis of the purified protein by size-exclusion chromatography suggests that gpNu3 is highly asymmetric, which contributes to an abnormally large Stokes radius. The size-exclusion chromatography data further indicate that the protein self-associates in a concentration-dependent manner. This was confirmed by analytical ultracentrifugation studies, which reveal a monomer-dimer equilibrium (K_d,app ~ 50 μM) and an asymmetric protein structure at biologically relevant concentrations. Purified gpNu3 promotes the polymerization of gpE, the λ major capsid protein, into virus-like particles that possess a native-like procapsid morphology. The relevance of this work with respect to procapsid assembly in the complex double-stranded DNA viruses is discussed.     

Domain study of bacteriophage p22 coat protein and characterization of the capsid lattice transformation by hydrogen/deuterium exchange

Viral capsids are dynamic structures which undergo a series of structural transformations to form infectious viruses. The dsDNA bacteriophage P22 is used as a model system to study the assembly and maturation of icosahedral dsDNA viruses. The P22 procapsid, which is the viral capsid precursor, is assembled from coat protein with the aid of scaffolding protein. Upon DNA packaging, the capsid lattice expands and becomes a stable virion. Limited proteolysis and biochemical experiments indicated that the coat protein consists of two domains connected by a flexible loop. To investigate the properties and roles of the sub-domains, we have cloned them and initiated structure and function studies. The N-terminal domain, which is made up of 190 amino acid residues, is largely unstructured in solution, while the C-terminal domain, which consists of 239 amino acid residues, forms a stable non-covalent dimer. The N-terminal domain adopts additional structure in the context of the C-terminal domain which might form a platform on which the N-terminal domain can fold. The local dynamics of the coat protein in both procapsids and mature capsids was monitored by hydrogen/deuterium exchange combined with mass spectrometry. The exchange rate for C-terminal domain peptides was similar in both forms. However, the N-terminal domain was more flexible in the empty procapsid shells than in the mature capsids. The flexibility of the N-terminal domain observed in the solution persisted into the procapsid form, but was lost upon maturation. The loop region connecting the two domains exchanged rapidly in the empty procapsid shells, but more slowly in the mature capsids. The global stabilization of the N-terminal domain and the flexibility encoded in the loop region may be a key component of the maturation process.     

Characterization of subunit structural changes accompanying assembly of the bacteriophage P22 procapsid

P22 serves as a model for the assembly and maturation of icosahedral double-stranded DNA viruses. The viral capsid precursor, or procapsid, is assembled from 420 copies of a 47 kDa coat protein subunit (gp5) that is rich in beta-strand secondary structure. Maturation to the capsid, which occurs in vivo upon DNA packaging, is accompanied by shell expansion and a large increase in the level of protection against deuterium exchange of amide NH groups. Accordingly, shell maturation resembles the final step in protein folding, wherein domain packing and an exchange-protected core become more fully developed [Tuma, R., Prevelige, P. E., Jr., and Thomas, G. J., Jr. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9885-9890]. Here, we exploit recent advances in Raman spectroscopy to investigate the P22 coat protein subunit under conditions which stabilize the monomeric state, viz., in solution at very low concentrations. Under these conditions, the monomer exhibits an elongated shape, as demonstrated by small-angle X-ray scattering. Raman spectra allow the identification of conformation-sensitive marker bands of the monomer, as well as the characterization of NH exchange dynamics for comparison with procapsid and capsid shell assemblies. We show that procapsid assembly involves significant ordering of the predominantly beta-strand backbone. We propose that such ordering may mediate formation of the distinct subunit conformations required for assembly of a T = 7 icosahedral lattice. However, the monomer, like the subunit within the procapsid lattice, exhibits a moderate level of protection against low-temperature NH exchange, indicative of a nascent folding core. The environments and exchange characteristics of key side chains are also similar for the monomeric and procapsid subunits, and distinct from corresponding characteristics of the capsid subunit. The monomer thus represents a compact but metastable folding intermediate along the pathway to assembly of the procapsid and capsid.     

Conformational transformations in the protein lattice of phage P22 procapsids.

During the packaging of double-stranded DNA by bacterial viruses, the precursor procapsid loses its internal core of scaffolding protein and undergoes a substantial expansion to form the mature virion. Here we show that upon heating, purified P22 procapsids release their scaffolding protein subunits, and the coat protein lattice expands in the absence of any other cellular or viral components. Following these processes by differential scanning calorimetry revealed four different transitions that correlated with structural transitions in the coat protein shells. Exit of scaffolding protein from the procapsid occurred reversibly and just above physiological temperature. Expansion of the procapsid lattice, which was exothermic, occurred after the release of scaffolding protein. Partial denaturation of coat subunits within the intact shell structure was detected prior to the major endothermic event. This major endotherm occurred above 80 degrees C and represents particle breakage and irreversible coat protein denaturation. The results indicate that the coat subunits are designed to form a metastable precursor lattice, which appears to be separated from the mature lattice by a kinetic barrier.     

Mechanism of scaffolding-directed virus assembly suggested by comparison of scaffolding-containing and scaffolding-lacking P22 procapsids.

Assembly of certain classes of bacterial and animal viruses requires the transient presence of molecules known as scaffolding proteins, which are essential for the assembly of the precursor procapsid. To assemble a procapsid of the proper size, each viral coat subunit must adopt the correct quasiequivalent conformation from several possible choices, depending upon the T number of the capsid. In the absence of scaffolding protein, the viral coat proteins form aberrantly shaped and incorrectly sized capsids that cannot package DNA. Although scaffolding proteins do not form icosahedral cores within procapsids, an icosahedrally ordered coat/scaffolding interaction could explain how scaffolding can cause conformational differences between coat subunits. To identify the interaction sites of scaffolding protein with the bacteriophage P22 coat protein lattice, we have determined electron cryomicroscopy structures of scaffolding-containing and scaffolding-lacking procapsids. The resulting difference maps suggest specific interactions of scaffolding protein with only four of the seven quasiequivalent coat protein conformations in the T = 7 P22 procapsid lattice, supporting the idea that the conformational switching of a coat subunit is regulated by the type of interactions it undergoes with the scaffolding protein. Based on these results, we propose a model for P22 procapsid assembly that involves alternating steps in which first coat, then scaffolding subunits form self-interactions that promote the addition of the other protein. Together, the coat and scaffolding provide overlapping sets of binding interactions that drive the formation of the procapsid.     

Regulation of coat protein polymerization by the scaffolding protein of bacteriophage P22.

In the morphogenesis of double stranded DNA phages, a precursor protein shell empty of DNA is first assembled and then filled with DNA. The assembly of the correctly dimensioned precursor shell (procapsid) of Salmonella bacteriophage P22 requires the interaction of some 420 coat protein subunits with approximately 200 scaffolding protein subunits to form a double shelled particle with the scaffolding protein on the inside. In the course of DNA packaging, all of the scaffolding protein subunits exit from the procapsid and participate in further rounds of procapsid assembly (King and Casjens. 1974. Nature (Lond.). 251:112-119). To study the mechanism of shell assembly we have purified the coat and scaffolding protein subunits by selective dissociation of isolated procapsids. Both proteins can be obtained as soluble subunits in Tris buffer at near neutral pH. The coat protein sedimented in sucrose gradients as a roughly spherical monomer, while the scaffolding protein sedimented as if it were an elongated monomer. When the two proteins were mixed together in 1.5 M guanidine hydrochloride and dialyzed back to buffer at room temperature, procapsids formed which were very similar in morphology, sedimentation behavior, and protein composition to procapsids formed in vivo. Incubation of either protein alone under the same conditions did not yield any large structures. We interpret these results to mean that the assembly of the shell involves a switching of both proteins from their nonaggregating to their aggregating forms through their mutual interaction. The results are discussed in terms of the general problem of self-regulated assembly and the control of protein polymerization in morphogenesis.     

In vitro assembly of the herpes simplex virus procapsid: formation of small procapsids at reduced scaffolding protein concentration

The herpes simplex virus 1 capsid is formed in the infected cell nucleus by way of a spherical, less robust intermediate called the procapsid. Procapsid assembly requires the capsid shell proteins (VP5, VP19C, and VP23) plus the scaffolding protein, pre-VP22a, a major component of the procapsid that is not present in the mature virion. Pre-VP22a is lost as DNA is packaged and the procapsid is transformed into the mature, icosahedral capsid. We have employed a cell-free assembly system to examine the role of the scaffolding protein in procapsid formation. While other reaction components (VP5, VP19C, and VP23) were held constant, the pre-VP22a concentration was varied, and the resulting procapsids were analyzed by electron microscopy and SDS-polyacrylamide gel electrophoresis. The results demonstrated that while standard-sized (T = 16) procapsids with a measured diameter of approximately 100 nm were formed above a threshold pre-VP22a concentration, at lower concentrations procapsids were smaller. The measured diameter was approximately 78 nm and the predicted triangulation number was 9. No procapsids larger than the standard size or smaller than 78-nm procapsids were observed in appreciable numbers at any pre-VP22a concentration tested. SDS-polyacrylamide gel analyses indicated that small procapsids contained a reduced amount of scaffolding protein compared to the standard 100-nm form. The observations indicate that the scaffolding protein concentration affects the structure of nascent procapsids with a minimum amount required for assembly of procapsids with the standard radius of curvature and scaffolding protein content.     

Role of the scaffolding protein in P22 procapsid size determination suggested by T = 4 and T = 7 procapsid structures.

Assembly of bacteriophage P22 procapsids requires the participation of approximately 300 molecules of scaffolding protein in addition to the 420 coat protein subunits. In the absence of the scaffolding, the P22 coat protein can assemble both wild-type-size and smaller size closed capsids. Both sizes of procapsid assembled in the absence of the scaffolding protein have been studied by electron cryomicroscopy. These structural studies show that the larger capsids have T = 7 icosahedral lattices and appear the same as wild-type procapsids. The smaller capsids possess T = 4 icosahedral symmetry. The two procapsids consist of very similar penton and hexon clusters, except for an increased curvature present in the T = 4 hexon. In particular, the pronounced skewing of the hexons is conserved in both sizes of capsid. The T = 7 procapsid has a local non-icosahedral twofold axis in the center of the hexon and thus contains four unique quasi-equivalent coat protein conformations that are the same as those in the T = 4 procapsid. Models of how the scaffolding protein may direct these four coat subunit types into a T = 7 rather than a T = 4 procapsid are presented.     

Visualization of the maturation transition in bacteriophage P22 by electron cryomicroscopy

Large-scale conformational transitions are involved in the life-cycle of many types of virus. The dsDNA phages, herpesviruses, and adenoviruses must undergo a maturation transition in the course of DNA packaging to convert a scaffolding-containing precursor capsid to the DNA-containing mature virion. This conformational transition converts the procapsid, which is smaller, rounder, and displays a distinctive skewing of the hexameric capsomeres, to the mature virion, which is larger and more angular, with regular hexons. We have used electron cryomicroscopy and image reconstruction to obtain 15 A structures of both bacteriophage P22 procapsids and mature phage. The maturation transition from the procapsid to the phage results in several changes in both the conformations of the individual coat protein subunits and the interactions between neighboring subunits. The most extensive conformational transformation among these is the outward movement of the trimer clusters present at all strict and local 3-fold axes on the procapsid inner surface. As the trimer tips are the sites of scaffolding binding, this helps to explain the role of scaffolding protein in regulating assembly and maturation. We also observe DNA within the capsid packed in a manner consistent with the spool model. These structures allow us to suggest how the binding interactions of scaffolding and DNA with the coat shell may act to control the packaging of the DNA into the expanding procapsids.     

Molecular genetics of bacteriophage P22 scaffolding protein's functional domains

The assembly intermediates of the Salmonella bacteriophage P22 are well defined but the molecular interactions between the subunits that participate in its assembly are not. The first stable intermediate in the assembly of the P22 virion is the procapsid, a preformed protein shell into which the viral genome is packaged. The procapsid consists of an icosahedrally symmetric shell of 415 molecules of coat protein, a dodecameric ring of portal protein at one of the icosahedral vertices through which the DNA enters, and approximately 250 molecules of scaffolding protein in the interior. Scaffolding protein is required for assembly of the procapsid but is not present in the mature virion. In order to define regions of scaffolding protein that contribute to the different aspects of its function, truncation mutants of the scaffolding protein were expressed during infection with scaffolding deficient phage P22, and the products of assembly were analyzed. Scaffolding protein amino acids 1-20 are not essential, since a mutant missing them is able to fully complement scaffolding deficient phage. Mutants lacking 57 N-terminal amino acids support the assembly of DNA containing virion-like particles; however, these particles have at least three differences from wild-type virions: (i) a less than normal complement of the gene 16 protein, which is required for DNA injection from the virion, (ii) a fraction of the truncated scaffolding protein was retained within the virions, and (iii) the encapsidated DNA molecule is shorter than the wild-type genome. Procapsids assembled in the presence of a scaffolding protein mutant consisting of only the C-terminal 75 amino acids contained the portal protein, but procapsids assembled with the C-terminal 66 did not, suggesting portal recruitment function for the region about 75 amino acids from the C terminus. Finally, scaffolding protein amino acids 280 through 294 constitute its minimal coat protein binding site.     

The mechanism for producing two symmetries at the head-tail junction of bacteriophages: a hypothesis

Some double-stranded DNA bacteriophages consist of DNA packaged in a proteinaceous capsid. The capsid has a DNA-enclosing outer shell (head) attached to an external projection (tail). At the head-tail junction is a ring of subunits (connector) that has either six or twelve-fold rotational symmetry, and is joined to the head at an axis of the head's five-fold rotational symmetry. The head is made of subunits in either an icosahedral array or an array consisting of two icosahedral hemispheres separated by a cylinder of subunits. During infection of a host, the head with connector is assembled as a procapsid that subsequently packages DNA and joins a tail. The mechanism for producing two symmetries at the head-tail junction has in the past been an unsolved problem. The observation that the connector of bacteriophage T7 does not nucleate assembly of the outer shell of T7's icosahedral procapsid (P. Serwer and R. H. Watson [1982] J. Virol. 42, 595-601) places a constraint on a solution for the above problem. To solve the above problem for icosahedral procapsids, it is proposed here that: (a) assembly of the outer shell of procapsids is nucleated by a six-membered ring of hexameric aggregates of the major outer shell protein, (b) the connector is assembled in the center of this ring, (c) one of the hexamers dissociates from the ring, creating a five-membered ring and forcing the connector to the inside of the outer shell. A related mechanism is proposed for nucleation of the elongated procapsid of bacteriophage T4.     

The role of scaffolding proteins in the assembly of the small, single-stranded DNA virus phiX174.

An empty precursor particle called the procapsid is formed during assembly of the single-stranded DNA bacteriophage phiX174. Assembly of the phiX174 procapsid requires the presence of the two scaffolding proteins, D and B, which are structural components of the procapsid, but are not found in the mature virion. The X-ray crystallographic structure of a "closed" procapsid particle has been determined to 3.5 A resolution. This structure has an external scaffold made from 240 copies of protein D, 60 copies of the internally located B protein, and contains 60 copies of each of the viral structural proteins F and G, which comprise the shell and the 5-fold spikes, respectively. The F capsid protein has a similar conformation to that seen in the mature virion, and differs from the previously determined 25 A resolution electron microscopic reconstruction of the "open" procapsid, in which the F protein has a different conformation. The D scaffolding protein has a predominantly alpha-helical fold and displays remarkable conformational variability. We report here an improved and refined structure of the closed procapsid and describe in some detail the differences between the four independent D scaffolding proteins per icosahedral asymmetric unit, as well as their interaction with the F capsid protein. We re-analyze and correct the comparison of the closed procapsid with the previously determined cryo-electron microscopic image reconstruction of the open procapsid and discuss the major structural rearrangements that must occur during assembly. A model is proposed in which the D proteins direct the assembly process by sequential binding and conformational switching.     

Folding and stability of mutant scaffolding proteins defective in P22 capsid assembly.

Bacteriophage P22 scaffolding subunits are elongated molecules that interact through their C termini with coat subunits to direct icosahedral capsid assembly. The soluble state of the subunit exhibits a partially folded intermediate during equilibrium unfolding experiments, whose C-terminal domain is unfolded (Greene, B., and King, J. (1999) J. Biol. Chem. 274, 16135-16140). Four mutant scaffolding proteins exhibiting temperature-sensitive defects in different stages of particle assembly were purified. The purified mutant proteins adopted a similar conformation to wild type, but all were destabilized with respect to wild type. Analysis of the thermal melting transitions showed that the mutants S242F and Y214W further destabilized the C-terminal domain, whereas substitutions near the N terminus either destabilized a different domain or affected interactions between domains. Two mutant proteins carried an additional cysteine residue, which formed disulfide cross-links but did not affect the denaturation transition. These mutants differed both from temperature-sensitive folding mutants found in other P22 structural proteins and from the thermolabile temperature-sensitive mutants described for T4 lysozyme. The results suggest that the defects in these mutants are due to destabilization of domains affecting the weak subunit-subunit interactions important in the assembly and function of the virus precursor shell.     

Novel second-site suppression of a cold-sensitive defect in phage P22 procapsid assembly

The DNA packaging portal of the phage P22 procapsid is formed of 12 molecules of the 90,000 dalton gene 1 protein. The assembly of this dodecameric complex at a unique capsid vertex requires scaffolding subunits. The mechanism that ensures the location of the 12-fold symmetrical portal at only one of the 12 5-fold vertices of an icosahedral virus capsid presents a unique assembly problem, which, in some viruses, is solved by the portal also acting as initiator of procapsid assembly. Phage P22 procapsids, however, are formed in the absence of the portal protein. The 1-csH137 mutation prevents the incorporation of the portal protein into procapsids. In a mixed infection with cs+ phage, the mutant subunits are able to form functional portals, suggesting that the cold-sensitivity does not affect portal-portal interactions, but affects the interaction of portal subunits with some other molecular species involved in the initiation of portal assembly. Interestingly, the cs defect is suppressed by temperature-sensitive folding mutations at four sites in the P22 tailspike gene 9. The suppression is allele-specific; other tailspike tsf mutations fail to suppress the cs defect. Translation through a suppressor site is required for suppression. This observation is unexpected, since analysis of nonsense mutations in this gene indicates that it is not required for procapsid assembly. Examination of the nucleic acid sequences in the neighborhood of each of the suppressor sites shows significant sequence similarity with the scaffolding gene translational initiation region on the late message. This supports a previously proposed model, in which procapsid assembly is normally initiated in a region on the late messenger RNA that includes the gene 8 start site. By this model, the suppressor mutations may be acting through protein-RNA interactions, changing sequences that identify alternative or competing sites at which the mutant portal subunits may be organized for assembly into the differentiated vertex of the phage capsid.     

Cavity defects in the procapsid of bacteriophage P22 and the mechanism of capsid maturation

Bacteriophage P22 belongs to a family of double-stranded DNA viruses that share common morphogenetic features like DNA packaging into a procapsid precursor and maturation. Maturation involves cooperative expansion of the procapsid shell with concomitant lattice stabilization. The expansion is thought to be mediated by movement of two coat protein domains around a hinge. The metastable conformation of subunit within the procapsid lattice is considered to constitute a late folding intermediate. In order to understand the mechanism of expansion it is necessary to characterize the interactions stabilizing procapsid and mature capsid lattices, respectively. We employ pressure dissociation to compare subunit packing within the procapsid and expanded lattice. Procapsid shells contain larger cavities than the expanded shells, presumably due to polypeptide packing defects. These defects contribute to the metastable nature of the procapsid lattice and are cured during expansion. Improved packing contributes to the increased stability of the expanded shell. Comparison of two temperature-sensitive folding (tsf) mutants of coat protein (T294I and W48Q) with wild-type coat revealed that both mutations markedly destabilized the procapsid shell and yet had little effect on relative stability of the monomeric subunit. Thus, the regions affected by these packing defects constitute subunit interfaces of the procapsid shell. The larger activation volume of pressure dissociation observed for both T294I and W48Q indicates that the decreased stability of these particles is due to increase of cavity defects. These defects in the procapsid lattice are cured upon expansion suggesting that the intersubunit contacts affected by tsf mutations are absent or rearranged in the mature shell. The energetics of the in vitro expansion reaction also suggests that entropic stabilization contributes to the large free energy barrier for expansion.     

The Prohead-I structure of bacteriophage HK97: implications for scaffold-mediated control of particle assembly and maturation

Virus capsid assembly requires recruiting and organizing multiple copies of protein subunits to form a closed shell for genome packaging that leads to infectivity. Many viruses encode scaffolding proteins to shift the equilibrium toward particle formation by promoting intersubunit interactions and stabilizing assembly intermediates. Bacteriophage HK97 lacks an explicit scaffolding protein, but the capsid protein (gp5) contains a scaffold-like N-terminal segment termed the delta domain. When gp5 is expressed in Escherichia coli, the delta domain guides 420 copies of the subunit into a procapsid with T = 7 laevo icosahedral symmetry named Prohead-I. Prohead-I can be disassembled and reassembled under mild conditions and it cannot mature further. When the virally encoded protease (gp4) is coexpressed with gp5, it is incorporated into the capsid and digests the delta domain followed by autoproteolysis to produce the metastable Prohead-II. Prohead-I^+P was isolated by coexpressing gp5 and an inactive mutant of gp4. Prohead-I and Prohead-I^+P were compared by biochemical methods, revealing that the inactive protease stabilized the capsid against disassembly by chemical or physical stress. The crystal structure of Prohead-I^+P was determined at 5.2 Å resolution, and distortions were observed in the subunit tertiary structures similar to those observed previously in Prohead-II. Prohead-I^+P differed from Prohead-II due to the presence of the delta domain and the resulting repositioning of the N-arms, explaining why Prohead-I can be reversibly dissociated and cannot mature. Low-resolution X-ray data enhanced the density of the relatively dynamic delta domains, revealing their quaternary arrangement and suggesting how they drive proper assembly.     

Quasi-atomic model of bacteriophage t7 procapsid shell: insights into the structure and evolution of a basic fold

The existence of similar folds among major structural subunits of viral capsids has shown unexpected evolutionary relationships suggesting common origins irrespective of the capsids' host life domain. Tailed bacteriophages are emerging as one such family, and we have studied the possible existence of the HK97-like fold in bacteriophage T7. The procapsid structure at approximately 10 A resolution was used to obtain a quasi-atomic model by fitting a homology model of the T7 capsid protein gp10 that was based on the atomic structure of the HK97 capsid protein. A number of fold similarities, such as the fitting of domains A and P into the L-shaped procapsid subunit, are evident between both viral systems. A different feature is related to the presence of the amino-terminal domain of gp10 found at the inner surface of the capsid that might play an important role in the interaction of capsid and scaffolding proteins.