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.     

Polyhead formation in phage P22 pinpoints a region in coat protein required for conformational switching

Eighteen single amino acid substitutions in phage P22 coat protein cause temperature-sensitive folding defects (tsf). Three intragenic global suppressor (su) substitutions (D163G, T166I and F170L), localized to a flexible loop, rescue the folding of several tsf coat proteins. Here we investigate the su substitutions in the absence of the original tsf substitutions. None of the su variant coat proteins displayed protein folding defects. Individual su substitutions had little effect on phage production in vivo; yet double and triple combinations resulted in a cold-sensitive (cs) phenotype, consistent with a defect in assembly. During virus assembly and maturation, conformational switching of capsid subunits is required when chemically identical capsid subunits form an icosahedron. Analysis of double- and triple-su phage-infected cell lysates by negative-stain electron microscopy reveals an increase in aberrant structures at the cs temperature. In vitro assembly of F170L coat protein causes production of polyheads, never seen before in phage P22. Purified procapsids composed of all of the su coat proteins showed defects in expansion, which mimics maturation in vitro. Our results suggest that a previously identified surface-exposed loop in coat protein is critical in conformational switching of subunits during both procapsid assembly and maturation.     

Molecular assembly and structure of the bacteriophage T4 tail

The tail of bacteriophage T4 undergoes large structural changes upon infection while delivering the phage genome into the host cell. The baseplate is located at the distal end of the contractile tail and plays a central role in transmitting the signal to the tail sheath that the tailfibers have been adsorbed by a host bacterium. This then triggers the sheath contraction. In order to understand the mechanism of assembly and conformational changes of the baseplate upon infection, we have determined the structure of an in vitro assembled baseplate through the three-dimensional reconstruction of cryo-electron microscopy images to a resolution of 3.8 Å from electron micrographs. The atomic structure was fitted to the baseplate structure before and after sheath contraction in order to elucidate the conformational changes that occur after bacteriophage T4 has attached itself to a cell surface. The structure was also used to investigate the protease digestion of the assembly intermediates and the mutation sites of the tail genes, resulting in a number of phenotypes.     

Assembly of the tail of bacteriophage T4

The protein products of at least 21 phage genes are needed for the formation of the tail of bacteriophage T4. Cells infected with amber mutants defective in these genes are blocked in the assembly process. By characterizing the intermediate structures and unassembled proteins accumulating in mutant-infected cells, we have been able to delineate most of the gene-controlled steps in tail assembly. Both the organized structures and unassembled proteins serve as precursors for in vitro tail assembly. We review here studies on the initiation, polymerization, and termination of the tail tube and contractile sheath and the genetic control of these processes. These studies make clear the importance of the baseplate; if baseplate formation is blocked (by mutation) the tube and sheath subunits remain essentially unaggregated, in the form of soluble subunits. Seventeen of the 21 tail genes specify proteins involved in baseplate assembly. The genes map contiguously in two separate clusters, one of nine genes and the other of eight genes. Recent studies show that the hexagonal baseplate is the end-product of two independent subassembly pathways. The proteins of the first gene cluster interact to form a structure which probably represents one-sixth of the outer radius. The products of the other gene cluster interact to form the central part of the baseplate. Most of the phage tail precursor proteins appear to be synthesized in a non-aggregating form; they are converted to a reactive form upon incorporation into preformed substrate complexes, without proteolytic cleavage. Thus reactive sites are limited to growing structures.     

The role of structural changes in T4 bacteriophage tail proteins

Conformational changes in bacteriophage tail proteins after heating and ionic strength alteration leading to dissociation of tail sheath have been studied using protein fluorescence, differential scanning microcalorimetry and electron microscopy methods. Autonomous structural changes in tube-baseplate proteins have been revealed. They take place under the same conditions as those which release the bonds holding the sheath protein subunits to those of the tube in isolated sheathed tails. The conformational changes in the tube-baseplates are reversible similarly to the process of assembly and disassembly of the extended sheath. Morphological changes in the tube have been found at the temperature above the transition registered by protein fluorescence but not by calorimetry. This suggests that revealed spectral alterations reflect changes in quaternary structure of tail tube in particular.     

The Type VI Secretion TssEFGK-VgrG Phage-Like Baseplate Is Recruited to the TssJLM Membrane Complex via Multiple Contacts and Serves As Assembly Platform for Tail Tube/Sheath Polymerization

The Type VI secretion system (T6SS) is a widespread weapon dedicated to the delivery of toxin proteins into eukaryotic and prokaryotic cells. The 13 T6SS subunits assemble a cytoplasmic contractile structure anchored to the cell envelope by a membrane-spanning complex. This structure is evolutionarily, structurally and functionally related to the tail of contractile bacteriophages. In bacteriophages, the tail assembles onto a protein complex, referred to as the baseplate, that not only serves as a platform during assembly of the tube and sheath, but also triggers the contraction of the sheath. Although progress has been made in understanding T6SS assembly and function, the composition of the T6SS baseplate remains mostly unknown. Here, we report that six T6SS proteins–TssA, TssE, TssF, TssG, TssK and VgrG–are required for proper assembly of the T6SS tail tube, and a complex between VgrG, TssE,-F and-G could be isolated. In addition, we demonstrate that TssF and TssG share limited sequence homologies with known phage components, and we report the interaction network between these subunits and other baseplate and tail components. In agreement with the baseplate being the assembly platform for the tail, fluorescence microscopy analyses of functional GFP-TssF and TssK-GFP fusion proteins show that these proteins assemble stable and static clusters on which the sheath polymerizes. Finally, we show that recruitment of the baseplate to the apparatus requires initial positioning of the membrane complex and contacts between TssG and the inner membrane TssM protein.     

Structure–Function Analysis of the C-Terminal Domain of the Type VI Secretion TssB Tail Sheath Subunit

The type VI secretion system (T6SS) is a specialized macromolecular complex dedicated to the delivery of protein effectors into both eukaryotic and bacterial cells. The general mechanism of action of the T6SS is similar to the injection of DNA by contractile bacteriophages. The cytoplasmic portion of the T6SS is evolutionarily, structurally and functionally related to the phage tail complex. It is composed of an inner tube made of stacked Hcp hexameric rings, engulfed within a sheath and built on a baseplate. This sheath undergoes cycles of extension and contraction, and the current model proposes that the sheath contraction propels the inner tube toward the target cell for effector delivery. The sheath comprises two subunits: TssB and TssC that polymerize under an extended conformation. Here, we show that isolated TssB forms trimers, and we report the crystal structure of a C-terminal fragment of TssB. This fragment comprises a long helix followed by a helical hairpin that presents surface-exposed charged residues. Site-directed mutagenesis coupled to functional assay further showed that these charges are required for proper assembly of the sheath. Positioning of these residues in the extended T6SS sheath structure suggests that they may mediate contacts with the baseplate.     

Morphogenesis of the T4 tail and tail fibers

Remarkable progress has been made during the past ten years in elucidating the structure of the bacteriophage T4 tail by a combination of three-dimensional image reconstruction from electron micrographs and X-ray crystallography of the components. Partial and complete structures of nine out of twenty tail structural proteins have been determined by X-ray crystallography and have been fitted into the 3D-reconstituted structure of the "extended" tail. The 3D structure of the "contracted" tail was also determined and interpreted in terms of component proteins. Given the pseudo-atomic tail structures both before and after contraction, it is now possible to understand the gross conformational change of the baseplate in terms of the change in the relative positions of the subunit proteins. These studies have explained how the conformational change of the baseplate and contraction of the tail are related to the tail's host cell recognition and membrane penetration function. On the other hand, the baseplate assembly process has been recently reexamined in detail in a precise system involving recombinant proteins (unlike the earlier studies with phage mutants). These experiments showed that the sequential association of the subunits of the baseplate wedge is based on the induced-fit upon association of each subunit. It was also found that, upon association of gp53 (gene product 53), the penultimate subunit of the wedge, six of the wedge intermediates spontaneously associate to form a baseplate-like structure in the absence of the central hub. Structure determination of the rest of the subunits and intermediate complexes and the assembly of the hub still require further study.     

Dissection of the TssB-TssC Interface during Type VI Secretion Sheath Complex Formation

The Type VI secretion system (T6SS) is a versatile machine that delivers toxins into either eukaryotic or bacterial cells. At a molecular level, the T6SS is composed of a membrane complex that anchors a long cytoplasmic tubular structure to the cell envelope. This structure is thought to resemble the tail of contractile bacteriophages. It is composed of the Hcp protein that assembles into hexameric rings stacked onto each other to form a tube similar to the phage tail tube. This tube is proposed to be wrapped by a structure called the sheath, composed of two proteins, TssB and TssC. It has been shown using fluorescence microscopy that the TssB and TssC proteins assemble into a tubular structure that cycles between long and short conformations suggesting that, similarly to the bacteriophage sheath, the T6SS sheath undergoes elongation and contraction events. The TssB and TssC proteins have been shown to interact and a specific α-helix of TssB is required for this interaction. Here, we confirm that the TssB and TssC proteins interact in enteroaggregative E. coli. We further show that this interaction requires the N-terminal region of TssC and the conserved α-helix of TssB. Using site-directed mutagenesis coupled to phenotypic analyses, we demonstrate that an hydrophobic motif located in the N-terminal region of this helix is required for interaction with TssC, sheath assembly and T6SS function.     

Multiple distinct assemblies reveal conformational flexibility in the small heat shock protein Hsp26

Small heat shock proteins are a superfamily of molecular chaperones that suppress protein aggregation and provide protection from cell stress. A key issue for understanding their action is to define the interactions of subunit domains in these oligomeric assemblies. Cryo-electron microscopy of yeast Hsp26 reveals two distinct forms, each comprising 24 subunits arranged in a porous shell with tetrahedral symmetry. The subunits form elongated, asymmetric dimers that assemble via trimeric contacts. Modifications of both termini cause rearrangements that yield a further four assemblies. Each subunit contains an N-terminal region, a globular middle domain, the alpha-crystallin domain, and a C-terminal tail. Twelve of the C termini form 3-fold assembly contacts which are inserted into the interior of the shell, while the other 12 C termini form contacts on the surface. Hinge points between the domains allow a variety of assembly contacts, providing the flexibility required for formation of supercomplexes with non-native proteins.     

Large-scale,pH-dependent,quaternary structure changes in an RNA virus capsid are reversible in the absence of subunit autoproteolysis

The assembly and maturation of the coat protein of a T=4, nonenveloped, single-stranded RNA virus, Nudaurelia capensis omega virus (N omega V), was examined by using a recombinant baculovirus expression system. At pH 7.6, the coat protein assembles into a stable particle called the procapsid, which is 450 A in diameter and porous. Lowering the pH to 5.0 leads to a concerted reorganization of the subunits into a 410-A-diameter particle called the capsid, which has no obvious pores. This conformational change is rapid but reversible until slow, autoproteolytic cleavage occurs in at least 15% of the subunits at the lower pH. In this report, we show that expression of subunits with replacement of Asn-570, which is at the cleavage site, with Thr results in assembly of particles with expected morphology but that are cleavage defective. The conformational change from procapsid to capsid is reversible in N570T mutant virus-like particles, in contrast to wild-type particles, which are locked into the capsid conformation after cleavage of the coat protein. The reexpanded procapsids display slightly different properties than the original procapsid, suggesting hysteretic effects. Because of the stability of the procapsid under near-neutral conditions and the reversible properties of the cleavage-defective mutant, N omega V provides an excellent model for the study of pH-induced conformational changes in macromolecular assemblies. Here, we identify the relationship between cleavage and the conformational change and propose a pH-dependent helix-coil transition that may be responsible for the structural rearrangement in N omega V.     

Type VI secretion and bacteriophage tail tubes share a common assembly pathway

The Type VI secretion system (T6SS) is a widespread macromolecular structure that delivers protein effectors to both eukaryotic and prokaryotic recipient cells. The current model describes the T6SS as an inverted phage tail composed of a sheath‐like structure wrapped around a tube assembled by stacked Hcp hexamers. Although recent progress has been made to understand T6SS sheath assembly and dynamics, there is no evidence that Hcp forms tubes in vivo. Here we show that Hcp interacts with TssB, a component of the T6SS sheath. Using a cysteine substitution approach, we demonstrate that Hcp hexamers assemble tubes in an ordered manner with a head‐to‐tail stacking that are used as a scaffold for polymerization of the TssB/C sheath‐like structure. Finally, we show that VgrG but not TssB/C controls the proper assembly of the Hcp tubular structure. These results highlight the conservation in the assembly mechanisms between the T6SS and the bacteriophage tail tube/sheath.     

Protein interactions in the assembly of the tail of bacteriophage T4

Protein interactions in the assembly of the baseplate have been investigated. The baseplate of the phage T4 tail consists of a hub and six wedges which surround the former. Both reversible and irreversible interactions were found. Reversible association includes gp5 and gp27 (gp: gene product) which form a complex in a pH-dependent manner and gp18 polymerization, i.e. the tail sheath formation depends on the ionic strength. These reversible interactions were followed by irreversible or tight binding which pulls the whole association reaction to complete the assembly. The wedge assembly is strictly ordered which means that if one of the seven wedge proteins is missing, the assembly proceeds to that point and the remaining molecules stay non-associated. The strictly sequential assembly pathway is suggested to be materialized by successive conformational change upon binding, which can be shown by proteolytic probe.     

A conformational switch in bacteriophage p22 portal protein primes genome injection

Double-stranded DNA (dsDNA) viruses such as herpesviruses and bacteriophages infect by delivering their genetic material into cells, a task mediated by a DNA channel called "portal protein." We have used electron cryomicroscopy to determine the structure of bacteriophage P22 portal protein in both the procapsid and mature capsid conformations. We find that, just as the viral capsid undergoes major conformational changes during virus maturation, the portal protein switches conformation from a procapsid to a mature phage state upon binding of gp4, the factor that initiates tail assembly. This dramatic conformational change traverses the entire length of the DNA channel, from the outside of the virus to the inner shell, and erects a large dome domain directly above the DNA channel that binds dsDNA inside the capsid. We hypothesize that this conformational change primes dsDNA for injection and directly couples completion of virus morphogenesis to a new cycle of infection.     

Structural basis for the assembly and gate closure mechanisms of the Mycobacterium tuberculosis 20S proteasome

Mycobacterium tuberculosis (Mtb) possesses a proteasome system analogous to the eukaryotic ubiquitin‐proteasome pathway. Mtb requires the proteasome to resist killing by the host immune system. The detailed assembly process and the gating mechanism of Mtb proteasome have remained unknown. Using cryo‐electron microscopy and X‐ray crystallography, we have obtained structures of three Mtb proteasome assembly intermediates, showing conformational changes during assembly, and explaining why the β‐subunit propeptide inhibits rather than promotes assembly. Although the eukaryotic proteasome core particles close their protein substrate entrance gates with different amino terminal peptides of the seven α‐subunits, it has been unknown how a prokaryotic proteasome might close the gate at the symmetry axis with seven identical peptides. We found in the new Mtb proteasome crystal structure that the gate is tightly sealed by the seven identical peptides taking on three distinct conformations. Our work provides the structural bases for assembly and gating mechanisms of the Mtb proteasome.     

Scaffolding proteins altered in the ability to perform a conformational switch confer dominant lethal assembly defects

In the phiX174 procapsid crystal structure, 240 external scaffolding protein D subunits form 60 pairs of asymmetric dimers, D(1)D(2) and D(3)D(4), in a non-quasi-equivalent structure. To achieve this arrangement, alpha-helix 3 assumes two different conformations: (i) kinked 30 degrees at glycine residue 61 in subunits D(1) and D(3) and (ii) straight in subunits D(2) and D(4). Substitutions for G61 may inhibit viral assembly by preventing the protein from achieving its fully kinked conformation while still allowing it to interact with other scaffolding and structural proteins. Mutations designed to inhibit conformational switching in alpha-helix 3 were introduced into a cloned gene, and expression was demonstrated to inhibit wild-type morphogenesis. The severity of inhibition appears to be related to the size of the substituted amino acid. For infections in which only the mutant protein is present, morphogenesis does not proceed past the first step that requires the wild-type external scaffolding protein. Thus, mutant subunits alone appear to have little or no morphogenetic function. In contrast, assembly in the presence of wild-type and mutant subunits is blocked prematurely, before D protein is required in a wild-type infection, or channeled into an off-pathway reaction. These data suggest that the wild-type protein transports the inhibitory protein to the pathway. Viruses resistant to the lethal dominant proteins were isolated, and mutations were mapped to the coat and internal scaffolding proteins. The affected amino acids cluster in the atomic structure and may act to exclude mutant subunits from occupying particular positions atop pentamers of the viral coat protein.     

TssK Is a Trimeric Cytoplasmic Protein Interacting with Components of Both Phage-like and Membrane Anchoring Complexes of the Type VI Secretion System

The Type VI secretion system (T6SS) is a macromolecular machine that mediates bacteria-host or bacteria-bacteria interactions. The T6SS core apparatus assembles from 13 proteins that form two sub-assemblies: a phage-like complex and a trans-envelope complex. The Hcp, VgrG, TssE, and TssB/C subunits are structurally and functionally related to components of the tail of contractile bacteriophages. This phage-like structure is thought to be anchored to the membrane by a trans-envelope complex composed of the TssJ, TssL, and TssM proteins. However, how the two sub-complexes are connected remains unknown. Here we identify TssK, a protein that establishes contacts with the two T6SS sub-complexes through direct interactions with TssL, Hcp, and TssC. TssK is a cytoplasmic protein assembling trimers that display a three-armed shape, as revealed by TEM and SAXS analyses. Fluorescence microscopy experiments further demonstrate the requirement of TssK for sheath assembly. Our results suggest a central role for TssK by linking both complexes during T6SS assembly.     

Roles for the troponin tail domain in thin filament assembly and regulation. A deletional study of cardiac troponin T

Striated muscle contraction is regulated by Ca2+ binding to troponin, which has a globular domain and an elongated tail attributable to the NH2-terminal portion of the bovine cardiac troponin T (TnT) subunit. Truncation of the bovine cardiac troponin tail was investigated using recombinant TnT fragments and subunits TnI and TnC. Progressive truncation of the troponin tail caused progressively weaker binding of troponin-tropomyosin to actin and of troponin to actin-tropomyosin. A sharp drop-off in affinity occurred with NH2-terminal deletion of 119 rather than 94 residues. Deletion of 94 residues had no effect on Ca2+-activation of the myosin subfragment 1-thin filament MgATPase rate and did not eliminate cooperative effects of Ca2+ binding. Troponin tail peptide TnT1-153 strongly promoted tropomyosin binding to actin in the absence of TnI or TnC. The results show that the anchoring function of the troponin tail involves interactions with actin as well as with tropomyosin and has comparable importance in the presence or absence of Ca2+. Residues 95-153 are particularly important for anchoring, and residues 95-119 are crucial for function or local folding. Because striated muscle regulation involves switching among the conformational states of the thin filament, regulatory significance for the troponin tail may arise from its prominent contribution to the protein-protein interactions within these conformations.     

The role of subunit hinges and molecular "switches" in the control of viral capsid polymorphism

The coat protein (CP) of cowpea chlorotic mottle virus assembles exclusively into a T=3 capsid in vivo and, under proper conditions, in vitro. The N-terminal domain of CP has been implicated in proper assembly and was viewed as a required switch for mediating hexamer and pentamer formation in T=3 assembly. We observed that a mutant CP lacking most of the N-terminal domain, NDelta34, assembles, in vitro, into statistically predictable numbers of: native-like T=3 capsids of 90 dimers; "T=2" capsids of 60 dimers; T=1 capsids of 30 dimers. We generated cryo-EM image reconstructions of each form and built pseudo-atomic models based on the subunits from the crystal structure of plant-derived T=3 virus allowing a detailed comparison of stabilizing interactions in the three assemblies. The statistical nature of the distribution of assembly products and the observed structures indicates that the N-terminus of CP is not a switch that is required to form the proper ratio of hexamers and pentamers for T=3 assembly; rather, it biases the direction of assembly to T=3 particles from the possibilities available to NDelta34 through flexible dimer hinges and variations in subunit contacts. Our results are consistent with a pentamer of dimers (PODs) nucleating assembly in all cases but subunit dimers can be added with different trajectories that favor specific T=3 or T=1 global particle geometries. Formation of the "T=2" particles appears to be fundamentally different in that they not only nucleate with PODs, but assembly propagates by the addition of mostly, if not exclusively PODs generating an entirely new subunit interface in the process. These results show that capsid geometry is flexible and may readily adapt to new requirements as the virus evolves.     

Assembly of tobacco mosaic virus in vitro. Improved model for the elongation process by protein subunits.

The in vitro assembly reaction of tobacco mosaic virus (TMV), especially the elongation process of partially reconstituted RNA (PRR) by protein subunits, was observed by electron microscopy. After addition of TMV-protein subunits, the PRR appeared as rods with a clump at one end, believed to be a complex between added protein subunits and the RNA tail protruding from PRR. The subunits entrapped on the RNA tails in the forms of clumps were progressively incorporated into the growing rods on incubation, ending with the formation of completely reconstituted rods. The clumps were also observed after addition of cucumber green mottle mosaic virus (CGMMV) protein subunits to rods partially reconstituted from RNA and TMV-protein. In this case, the protein subunits, seen as clumps, did not become incorporated to form elongating rods. An improved model for the elongation of TMV rods is proposed. The elongation process is composed of two steps, with the first step being the interaction of protein subunits with the RNA tail protruding from the growing rod. Any protein having a specific binding site for TMV-rna, not limited to TMV-protein, will react in the first step. The second step is the incorporation of the protein on the RNA tail into a rod-shaped structure, with consequent elongation of the growing rod. It appears that only protein homologous with that in the partially reconstituted rods can partake in the second step.