Robert L. Duda

Structural transitions during bacteriophage HK97 head assembly

Bacteriophage HK97 builds its head shell from a 42 kDa major head protein, but neither this 42 kDa protein nor its processed, 31 kDa form is found in the mature head. Instead, each of the major head-protein subunits is covalently cross-linked into oligomers of five, six or more by a protein cross-linking reaction that occurs both in vivo and in vitro. Mutants that block prohead maturation lead to the accumulation of one of two types of proheads, termed Prohead I and Prohead II. Prohead I is assembled from about 415 copies of the 42 kDa (384 amino acids) protein subunit and accumulates in infections by mutant amU4. Following assembly, the N-terminal 102 amino acids of each subunit are removed, leaving a prohead shell constructed of 31 kDa subunits, called Prohead II, which accumulates in infections by mutant amC2. During DNA packaging, when the prohead shell expands, all of the head protein subunits become covalently cross-linked to other subunits. Purified Prohead II (or, less completely, Prohead I) becomes cross-linked in vitro in response to any of a number of conditions that induce shell expansion, including conditions commonly used for protein analysis. In vitro cross-linking occurs efficiently in the absence of added cofactors of enzymes, and we propose that cross-linking is catalyzed by shell subunits themselves. Shell expansion is easily monitored by observing a decrease in electrophoretic mobility of Prohead II in agarose gels. Using the mobility shift in agarose gel to monitor expansion and SDS/gel electrophoresis to monitor cross-linking in vitro, we find that expansion precedes and is required for cross-linking, and we propose that expansion triggers the cross-linking reaction. Comparison of peptides isolated from Prohead II and in vitro cross-linked Prohead II shows a single altered major cross-link peptide in which a lysine, originating from lysine169 of the protein sequence, is linked to asparagine356, presumably derived from the neighboring subunit. Examination of the cross-link-containing peptide by mass spectrometry shows that the cross-link bond is an amide between the side-chains of the lysine and the asparagine residues.

Protein Chainmail: Catenated Protein in Viral Capsids

The capsid shells of bacteriophage HK97 and several other phages contain polypeptides that are covalently linked into complexes so large that they do not enter polyacrylamide gels after denaturation. The enormous apparent size of these protein complexes in HK97 derives from a novel protein topology. HK97 subunits cross-link via isopeptide bonds into oligomers that are closed rings of five or six members. However, polypeptides from neighboring pentamer and hexamer rings intertwine before the covalent cross-links form. As a result, adjacent protein rings catenate into a network similar to chainmail armor. In vitro linking and unlinking experiments provide strong support for the chainmail model, which explains the unusual properties of these bacteriophages and may apply to other macromolecular structures.

Genetic basis of bacteriophage HK97 prohead assembly

We report studies to determine which bacteriophage genes are required for assembly of phage HK97 proheads and what roles they play. We identify the gene encoding the major capsid protein of phage HK97 and report its DNA sequence, together with the DNA sequences of the two genes immediately upstream from it. When the capsid protein is expressed from a plasmid in the absence of other phage-encoded proteins, it assembles, with good efficiency and accuracy into prohead-like structures composed of the unprocessed 42 kDa capsid protein. No separately encoded scaffolding protein is required for this assembly. If the 25 kDa product of the next gene upstream is co-expressed with the capsid protein, the prohead structures that are produced undergo the normal morphogenetic cleavage, which removes 102 amino acids from the N terminus of each subunit, leaving 31 kDa subunits. The 25 kDa protein is therefore probably a phage-encoded protease. The third gene, upstream from the protease gene, encodes the portal protein. Presence of the portal protein is not required for assembly of the capsid protein. Analysis of the phenotypes of four single amino acid-substitution mutants in the capsid-protein gene leads to several insights into the functions of the capsid protein and its interactions with the putative protease.

An unexpected twist in viral capsid maturation

Lambda-like double-stranded (ds) DNA bacteriophage undergo massive conformational changes in their capsid shell during the packaging of their viral genomes. Capsid shells are complex organizations of hundreds of protein subunits that assemble into intricate quaternary complexes that ultimately are able to withstand over 50 atm of pressure during genome packaging. The extensive integration between subunits in capsids requires the formation of an intermediate complex, termed a procapsid, from which individual subunits can undergo the necessary refolding and structural rearrangements needed to transition to the more stable capsid. Although various mature capsids have been characterized at atomic resolution, no such procapsid structure is available for a dsDNA virus or bacteriophage. Here we present a procapsid X-ray structure at 3.65 Å resolution, termed prohead II, of the lambda-like bacteriophage HK97, the mature capsid structure of which was previously solved to 3.44 Å (ref. 2). A comparison of the two largely different capsid forms has unveiled an unprecedented expansion mechanism that describes the transition. Crystallographic and hydrogen/deuterium exchange data presented here demonstrate that the subunit tertiary structures are significantly different between the two states, with twisting and bending motions occurring in both helical and β-sheet regions. We also identified subunit interactions at each three-fold axis of the capsid that are maintained throughout maturation. The interactions sustain capsid integrity during subunit refolding and provide a fixed hinge from which subunits undergo rotational and translational motions during maturation. Previously published calorimetric data of a closely related bacteriophage, P22, showed that capsid maturation was an exothermic process that resulted in a release of 90 kJ mol-1 of energy. We propose that the major tertiary changes presented in this study reveal a structural basis for an exothermic maturation process probably present in many dsDNA bacteriophage and possibly viruses such as herpesvirus, which share the HK97 subunit fold.

Genome organization and characterization of mycobacteriophage Bxb1

Mycobacteriophage Bxb1 is a temperate phage of Mycobacterium smegmatis. The morphology of Bxb1 particles is similar to that of mycobacteriophages L5 and D29, although Bxb1 differs from these phages in other respects. First, it is heteroimmune with L5 and efficiently forms plaques on an L5 lysogen. Secondly, it has a different host range and fails to infect slow‐growing mycobacteria, using a receptor system that is apparently different from that of L5 and D29. Thirdly, it is the first mycobacteriophage to be described that forms a large prominent halo around plaques on a lawn of M. smegmatis. The sequence of the Bxb1 genome shows that it possesses a similar overall organization to the genomes of L5 and D29 and shares weak but detectable DNA sequence similarity to these phages within the structural genes. However, Bxb1 uses a different system of integration and excision, a repressor with different specificity to that of L5 and encodes a large number of novel gene products including several with enzymatic functions that could degrade or modify the mycobacterial cell wall.

Bacteriophage HK97 Head Assembly: A Protein Ballet

Publisher Summary When biologists first began to think about how a structure like a bacteriophage virion might assemble from its macromolecular parts, it seemed to be mostly a problem of mustering enough specific bonding interactions among all of the participating components and giving them time to find one another and stick together. The individual proteins were generally seen as rigid and unchanging objects, much like the bricks with which one might assemble a house. The overview presented in this chapter is a much more dynamic one, especially at the level of the conformational flexibility of individual proteins. Considering only the structural transitions that is characterized in HK97 head shell assembly, the conformational versatility can be ascribe to the head subunit is quite remarkable. The subunit initially assembles alternatively into two kinds of oligomers, pentamers, and hexamers, and those assemble into shells. At some point during that process the hexamers become dramatically skewed, presumably requiring additional, and substantially different conformations and intersubunit interactions. The chapter presents the full performance of the HK97 ballet as viewed through the optics, compares the HK97 program with those of other viruses and other proteins, and speculates on the causes and purposes of the individual steps and what they reveal about how proteins work. The mechanisms used by HK97 to control head shell assembly and maturation appear in many ways to differ from those used by other phages, but these differences can be reconciled with a set of common themes that govern the action and function of scaffolds, proteolysis, and expansion in bacteriophage capsid assembly.

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

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

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.

Shared architecture of bacteriophage SPO1 and herpesvirus capsids

Document S1. One figure and Supplemental Experimental ProceduresxDownload (.13 MB ) Document S1. One figure and Supplemental Experimental Procedures

Structure and Energetics of Encapsidated DNA in Bacteriophage HK97 Studied by Scanning Calorimetry and Cryo-electron Microscopy

Encapsidation of duplex DNA by bacteriophages represents an extreme case of genome condensation, reaching near-crystalline concentrations of DNA. The HK97 system is well suited to study this phenomenon in view of the detailed knowledge of its capsid structure. To characterize the interactions involved, we combined calorimetry with cryo-electron microscopy and native gel electrophoresis. We found that, as in other phages, HK97 DNA is organized in coaxially wound nested shells. When DNA-filled capsids (heads) are scanned in buffer containing 1 mM Mg(2+), DNA melting and capsid denaturation both contribute to the complex thermal profile between 82 degrees C and 96 degrees C. In other conditions (absence of Mg(2+) and lower ionic strength), DNA melting shifts to lower temperatures and the two events are resolved. Heads release their DNA at temperatures well below the onset of DNA melting or capsid denaturation. We suggest that, on heating, the internal pressure increases, causing the DNA to exit-probably via the portal vertex-while the capsid, although largely intact, sustains local damage that leads to an earlier onset of thermal denaturation. Heads differ structurally from empty capsids in the curvature of their protein shell, a change attributable to outwards pressure exerted by the DNA. We propose that this transition is sensed by the portal that is embedded in the capsid wall, whereupon the structure of the portal and its interactions with terminase, the packaging enzyme, are altered, thus signaling that packaging is at or approaching completion.