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J Biol Chem. 2016 Jan 1;291(1):215-26. doi: 10.1074/jbc.M115.696260. Epub 2015 Nov 16.

Structural Plasticity of the Protein Plug That Traps Newly Packaged Genomes in Podoviridae Virions.

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From the Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
the Department of Biochemistry & Biophysics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania 19104.
the Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294.
the Division of Microbiology and Immunology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah 84112.
the Department of Pharmacology, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106, and.
From the Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, the Institute of Biomembranes and Bioenergetics, National Research Council, 70126 Bari, Italy


Bacterial viruses of the P22-like family encode a specialized tail needle essential for genome stabilization after DNA packaging and implicated in Gram-negative cell envelope penetration. The atomic structure of P22 tail needle (gp26) crystallized at acidic pH reveals a slender fiber containing an N-terminal "trimer of hairpins" tip. Although the length and composition of tail needles vary significantly in Podoviridae, unexpectedly, the amino acid sequence of the N-terminal tip is exceptionally conserved in more than 200 genomes of P22-like phages and prophages. In this paper, we used x-ray crystallography and EM to investigate the neutral pH structure of three tail needles from bacteriophage P22, HK620, and Sf6. In all cases, we found that the N-terminal tip is poorly structured, in stark contrast to the compact trimer of hairpins seen in gp26 crystallized at acidic pH. Hydrogen-deuterium exchange mass spectrometry, limited proteolysis, circular dichroism spectroscopy, and gel filtration chromatography revealed that the N-terminal tip is highly dynamic in solution and unlikely to adopt a stable trimeric conformation at physiological pH. This is supported by the cryo-EM reconstruction of P22 mature virion tail, where the density of gp26 N-terminal tip is incompatible with a trimer of hairpins. We propose the tail needle N-terminal tip exists in two conformations: a pre-ejection extended conformation, which seals the portal vertex after genome packaging, and a postejection trimer of hairpins, which forms upon its release from the virion. The conformational plasticity of the tail needle N-terminal tip is built in the amino acid sequence, explaining its extraordinary conservation in nature.


DNA packaging; bacteriophage; hydrogen-deuterium exchange; infection; protein folding; tail needle; trimer of hairpins; viral genome ejection; x-ray crystallography

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