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

ABSTRACT: 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.

SUBMITTER: Huang RK

PROVIDER: S-EPMC3075369 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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

Huang Rick K RK Khayat Reza R Lee Kelly K KK Gertsman Ilya I Duda Robert L RL Hendrix Roger W RW Johnson John E JE

Journal of molecular biology 20110127 3

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 ...[more]

PMID: 21276801

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Project description:A major question in mapping protein-ligand or protein-protein interactions in solution is to distinguish direct-binding interactions from long-range conformational changes at allosteric sites. We describe here the applicability of amide hydrogen deuterium exchange mass spectrometry (HDXMS) in addressing this important question using the bacteriophage HK97 capsid proteins' interactions with their processing protease. HK97 is a lambda-like dsDNA bacteriophage that is ideal for studies of particle assembly and maturation. Its capsid precursor protein is composed of two main regions, the scaffolding protein (?-domain, residues 2-103), and the coat subunit (residues 104-385), which spontaneously forms a mixture of hexamers and pentamers upon association. Activation of the viral protease, which occurs after particle assembly, is initiated by the protease mediated digestion of the scaffolding domains to yield Prohead-2. This irreversible step is obligatory for activation of the virus maturation pathway. Here we provide an "addendum" to our previous study of Prohead I and Prohead I+pro (a transient complex of Prohead I and the protease) where we investigated the interactions between the ? domain and the packaged protease using HDXMS. Our results revealed two sites on the ? domain: one set of contiguous peptides that showed decreased exchange at the protease binding site at early time points of deuterium labeling and another separate set of continuous peptides that showed decreased exchange at later time points. Even though this cannot reveal the time scales of molecular processes governing binding and allostery, we believe this differential pattern of exchange across deuteration times can allow spatial distinction between binding sites and long range conformational changes with allosteric implications. This partitioning can be discerned from the lag between noncontiguous regions on a protein showing maximal changes in deuterium exchange and highlights a powerful application of HDXMS in distinguishing direct binding in transient protein-protein interactions from allosteric changes.

Dataset Information

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

Publications

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

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