title: Crosslinking renders bacteriophage HK97 capsid maturation irreversible and effects an essential stabilization.
abstract: In HK97 capsid maturation, structural change ('expansion') is accompanied by formation of covalent crosslinks, connecting residue K169 in the 'E-loop' of each subunit with N356 on another subunit. We show by complementation experiments with the K169Y mutant, which cannot crosslink, that crosslinking is an essential function. The precursor Prohead-II passes through three expansion intermediate (EI) states en route to the end state, Head-II. We investigated the effects of expansion and crosslinking on stability by differential scanning calorimetry of wild-type and K169Y capsids. After expansion, the denaturation temperature (Tp) of K169Y capsids is slightly reduced, indicating that their thermal stability is not enhanced, but crosslinking effects a major stabilization (deltaTp, +11 degrees C). EI-II is the earliest capsid to form crosslinks. Cryo-electron microscopy shows that for both wild-type and K169Y EI-II, most E-loops are in the 'up' position, 30 A from the nearest N356: thus, crosslinking in EI-II represents capture of mobile E-loops in 'down' positions. At pH 4, most K169Y capsids remain as EI-II, whereas wild-type capsids proceed to EI-III, suggesting that crosslink formation drives maturation by a Brownian ratchet mechanism.
authors.affiliation: Laboratory of Molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, MD, USA.
authors.forename: Philip D
authors.initials: PD
authors.lastname: Ross
authors.forename: Naiqian
authors.initials: N
authors.lastname: Cheng
authors.forename: James F
authors.initials: JF
authors.lastname: Conway
authors.forename: Brian A
authors.initials: BA
authors.lastname: Firek
authors.forename: Roger W
authors.initials: RW
authors.lastname: Hendrix
authors.forename: Robert L
authors.initials: RL
authors.lastname: Duda
authors.forename: Alasdair C
authors.initials: AC
authors.lastname: Steven
mesh_terms: D001435:Bacteriophages / Q000502:physiology*; D002152:Calorimetry, Differential Scanning; D036022:Capsid Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000648:ultrastructure*; D020285:Cryoelectron Microscopy; D005816:Genetic Complementation Test; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D021122:Protein Subunits / Q000378:metabolism*; D014758:Viral Core Proteins / Q000378:metabolism; D019065:Virus Assembly / Q000502:physiology*
chemical_list: D036022:Capsid Proteins; D021122:Protein Subunits; D014758:Viral Core Proteins