Identification of subunit-subunit interactions in bacteriophage P22 procapsids by chemical cross-linking and mass spectrometry.
Viral capsids are dynamic structures which self-assemble and undergo a series of structural transformations to form infectious viruses. The dsDNA bacteriophage P22 is used as a model system to study the assembly and maturation of icosahedral dsDNA viruses. The P22 procapsid, which is the viral capsid precursor, is assembled from coat protein with the aid of scaffolding protein. Upon DNA packaging, the capsid lattice expands and becomes a stable virion. Chemical cross-linking analyzed by mass spectrometry was used to identify residue specific inter- and intra-subunit interactions in the P22 procapsids. All the intersubunit cross-links occurred between residues clustered in a loop region (residues 157-207) which was previously identified by mass spectrometry based on hydrogen/deuterium exchange and biochemical experiments. DSP and BS3 which have similar distance constraints (12 angstroms and 11.4 angstroms, respectively) cross-linked the same residues between two subunits in the procapsids (K183-K183), whereas DST, a shorter cross-linker, cross-linked lysine 175 in one subunit to lysine 183 in another subunit. The replacement of threonine with a cysteine at residue 182 immediately adjacent to the K183 cross-linking site resulted in slow spontaneous disulfide bond formation in the procapsids without perturbing capsid integrity, thus suggesting flexibility within the loop region and close proximity between neighboring loop regions. To build a detailed structure model, we have predicted the secondary structure elements of the P22 coat protein, and attempted to thread the prediction onto identified helical elements of cryoEM 3D reconstruction. In this model, the loop regions where chemical cross-linkings occurred correspond to the extra density (ED) regions which protrude upward from the outside of the capsids and face one another around the symmetry axes.
Kang
Sebyung
S
Department of Biochemistry & Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Hawkridge
Adam M
AM
Johnson
Kenneth L
KL
Muddiman
David C
DC
Prevelige
Peter E
PE
D019943:Amino Acid Substitution / Q000235:genetics; D017100:Bacteriophage P22 / Q000378:metabolism*; D002213:Capsid / Q000378:metabolism; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism*; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism*; D013912:Threonine / Q000737:chemistry / Q000235:genetics; D019065:Virus Assembly
D036022:Capsid Proteins; D003432:Cross-Linking Reagents; D021122:Protein Subunits; D013912:Threonine; D003545:Cysteine