The crystal structure of the double-stranded DNA bacteriophage HK97 mature empty capsid was determined at 3.6 angstrom resolution. The 660 angstrom diameter icosahedral particle contains 420 subunits with a new fold. The final capsid maturation step is an autocatalytic reaction that creates 420 isopeptide bonds between proteins. Each subunit is joined to two of its neighbors by ligation of the side-chain lysine 169 to asparagine 356. This generates 12 pentameric and 60 hexameric rings of covalently joined subunits that loop through each other, creating protein chainmail: topologically linked protein catenanes arranged with icosahedral symmetry. Catenanes have not been previously observed in proteins and provide a stabilization mechanism for the very thin HK97 capsid.

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A programmed translational frameshift similar to frameshifts in retroviral gag-pol genes and bacterial insertion elements was found to be strongly conserved in tail assembly genes of dsDNA phages and to be independent of sequence similarities. In bacteriophage lambda, this frameshift controls production of two proteins with overlapping sequences, gpG and gpGT, that are required for tail assembly. We developed bioinformatic approaches to identify analogous -1 frameshifting sites and experimentally confirmed our predictions for five additional phages. Clear evidence was also found for an unusual but analogous -2 frameshift in phage Mu. Frameshifting sites could be identified for most phages with contractile or noncontractile tails whose length is controlled by a tape measure protein. Phages from a broad spectrum of hosts spanning Eubacteria and Archaea appear to conserve this frameshift as a fundamental component of their tail assembly mechanisms, supporting the idea that their tail genes share a common, distant ancestry.

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Lambda-like dsDNA 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 1 . The extensive integration between subunits in capsids is unlikely to form in a single assembly step, therefore requiring 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. Though various mature capsids have been characterized at atomic resolution, no such procapsid structure is available for a dsDNA virus or bacteriophage that undergoes large scale conformational changes. We present a procapsid x-ray structure at 3.65Å resolution, termed Prohead II, of the lambda like bacteriophage HK97, whose mature capsid structure was previously solved to 3.44 Å 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 demonstrates 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 have also discovered conserved subunit interactions at each 3-fold of the virus capsid, from which capsid subunits maintain their integrity during refolding, facilitating the rotational and translational motions of maturation. Calormetric data of a closely related bacteriophage, P22, showed that capsid maturation was an exothermic process that resulted in a release of 90KJ/mol of energy 3 . We propose the major tertiary changes presented in this study reveal a structural basis for an exothermic maturation process likely present in many dsDNA Bacteriophage and possibly viruses such as Herpes which share the HK97 subunit fold 4 . Main TextHK97 is a favorable system for studying capsid maturation because capsid particles can be assembled in E. coli from the expression of just two viral gene products, gp4 (protease) and gp5 (capsid subunit), and maturation can be triggered and analyzed in vitro 2,5-8 . The 384-residue gp5 subunits assemble into hexameric and pentameric oligomers, termed capsomers, that first assemble to form the Prohead I capsid (P-I). The 17.7-Megadalton, T=7 laevo icosahedral particle contains 12 pentamers and 60 hexamers and encapsidates approximately 60 copies of the gp4 protease 9-11 . Prohead I particles can be made with either a defective protease or without protease and can be disassembled in vitro into free capsomers and than reassembled when exposed to specific chemical treatments 12 . When active gp4 is present, the particles spontaneously mature to the Prohead II (P-II) form following digestion of residues 2-103 from all subunits. The proteolytic fragment...