title: A free energy cascade with locks drives assembly and maturation of bacteriophage HK97 capsid.
abstract: We investigated the thermodynamic basis of HK97 assembly by scanning calorimetry and cryo-electron microscopy. This pathway involves self-assembly of hexamers and pentamers of the precursor capsid protein gp5 into procapsids; proteolysis of their N-terminal Delta-domains; expansion, a major conformational change; and covalent crosslinking. The thermal denaturation parameters convey the changes in stability at successive steps in assembly, and afford estimates of the corresponding changes in free energy. The procapsid represents a kinetically accessible local minimum of free energy. In maturation, it progresses to lower minima in a cascade punctuated by irreversible processes ("locks"), i.e. proteolysis and crosslinking, that lower kinetic barriers and prevent regression. We infer that Delta-domains not only guide assembly but also restrain the procapsid from premature expansion; their removal by proteolysis is conducive to initiating expansion and to its proceeding to completion. We also analyzed the mutant E219K, whose capsomers reassemble in vitro into procapsids with vacant vertices called "whiffleballs". E219K assemblies all have markedly reduced stability compared to wild-type gp5 (DeltaT(p) approximately -7 degrees C to -10 degrees C; where T(p) is the denaturation temperature). As the mutated residue is buried in the core of gp5, we attribute the observed reduction in stability to steric and electrostatic perturbations of the packing of side-chains in the subunit interior. To explain the whiffleball phenotype, we suggest that these effects propagate to the capsomer periphery in such a way as to differentially affect the stability or solubility of dissociated pentamers, leaving only hexamers to reassemble.
authors.affiliation: Laboratory of Molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, MD 20892, USA.
authors.forename: Philip D
authors.initials: PD
authors.lastname: Ross
authors.forename: James F
authors.initials: JF
authors.lastname: Conway
authors.forename: Naiqian
authors.initials: N
authors.lastname: Cheng
authors.forename: Lindsay
authors.initials: L
authors.lastname: Dierkes
authors.forename: Brian A
authors.initials: BA
authors.lastname: Firek
authors.forename: Roger W
authors.initials: RW
authors.lastname: Hendrix
authors.forename: Alasdair C
authors.initials: AC
authors.lastname: Steven
authors.forename: Robert L
authors.initials: RL
authors.lastname: Duda
mesh_terms: D001435:Bacteriophages / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D002152:Calorimetry, Differential Scanning; D002213:Capsid / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D036022:Capsid Proteins / Q000378:metabolism* / Q000648:ultrastructure; D020285:Cryoelectron Microscopy; D008958:Models, Molecular; D009154:Mutation; D017434:Protein Structure, Tertiary; D013816:Thermodynamics
chemical_list: D036022:Capsid Proteins