Structure and assembly of bacteriophages

Yizhi Tao, Purdue University

Abstract

Bacteriophages are usually complex molecular machines. Bacteriophages T4 and &phis;29 were investigated by combining the x-ray crystallography and three-dimensional (3-D) cryo-electron microscopy (cryo-EM) reconstruction methods. Both T4 and &phis;29 consist of a prolate head which encapsidates a dsDNA genome and a tail through which the DNA is ejected during infection. The tails and heads of these viruses are joined through a substructure called connector located at a special vertex. The structure of bacteriophage T4 fibritin deletion mutant E was solved to 2.2Å resolution by the multiple isomorphous replacement method (MIR). Fibritin forms the whiskers and collar complex substructure embracing the virion neck. The whiskers are both a molecular chaperone during assembly and a rudimentary environment-sensing device to prevent infection in certain adverse environments. Fibritin E consists of the last 119 residues of the w.t. protein, including three α-helical coiled coil segments linked by short loop structures and a small globular domain at the C-terminus. While introducing some flexibility, these insertion loops do not break the overall integrity of the coiled coil domain. The amino acid composition at the a and d positions of the coiled coil is not optimal for a trimer formation. Indeed, fibritin E coiled coil structure shows relatively loose “knobs-into-holes” packing. Moreover, there is mutational evidence that the globular C-terminal domain, which involves extensive hydrophobic and some polar interactions, is important for the correct self-assembly of fibritin. Presumably the strong interactions in the C-terminal domain stabilize the structure in the trimeric state. The &phis;29 virion of Bacillus subtilis and its empty prohead precursor as well as two isometric mutants have been studied by 3-D cryo-EM reconstruction. The head-tail connector, the central component of the DNA packaging machine, is visualized for the first time in situ . The connector, with 12- or 13-fold symmetry, appears to fit loosely into a pentameric vertex of the head, a symmetry mismatch that may be required to rotate the connector to package DNA. The prolate head of &phis;29 has 10 hexameric units in its cylindrical equatorial region, and 11 pentameric and 20 hexameric units comprise icosahedral end-caps with partial T = 3 quasi symmetry. Reconstruction of an emptied phage particle shows that the connector and neck/tail assembly undergo significant conformational changes upon ejection of DNA.

Degree

Ph.D.

Advisors

Rossmann, Purdue University.

Subject Area

Molecular biology|Biophysics

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