title: Structural Plasticity of the Protein Plug That Traps Newly Packaged Genomes in Podoviridae Virions.
abstract: 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.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
authors.forename: Anshul
authors.initials: A
authors.lastname: Bhardwaj
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
authors.forename: Rajeshwer S
authors.initials: RS
authors.lastname: Sankhala
authors.affiliation: the Department of Biochemistry & Biophysics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors.forename: Adam S
authors.initials: AS
authors.lastname: Olia
authors.affiliation: the Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294.
authors.forename: Dewey
authors.initials: D
authors.lastname: Brooke
authors.affiliation: the Division of Microbiology and Immunology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah 84112.
authors.forename: Sherwood R
authors.initials: SR
authors.lastname: Casjens
authors.affiliation: the Department of Pharmacology, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106, and.
authors.forename: Derek J
authors.initials: DJ
authors.lastname: Taylor
authors.affiliation: the Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294.
authors.forename: Peter E
authors.initials: PE
authors.lastname: Prevelige
authors.affiliation: 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 gino.cingolani@jefferson.edu.
authors.forename: Gino
authors.initials: G
authors.lastname: Cingolani
mesh_terms: D001435:Bacteriophages / Q000737:chemistry; D002942:Circular Dichroism; D020285:Cryoelectron Microscopy; D018360:Crystallography, X-Ray; D041961:Deuterium Exchange Measurement; D016679:Genome, Viral; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D016624:Negative Staining; D017902:Podoviridae / Q000235:genetics*; D055503:Protein Multimerization; D017376:Viral Tail Proteins / Q000737:chemistry* / Q000648:ultrastructure; D014771:Virion / Q000235:genetics*; D019065:Virus Assembly
chemical_list: D017376:Viral Tail Proteins
keywords: DNA packaging; bacteriophage; hydrogen-deuterium exchange; infection; protein folding; tail needle; trimer of hairpins; viral genome ejection; x-ray crystallography