Crosslinking renders bacteriophage HK97 capsid maturation irreversible and effects an essential stabilization.
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.
Ross
Philip D
PD
Laboratory of Molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, MD, USA.
Cheng
Naiqian
N
Conway
James F
JF
Firek
Brian A
BA
Hendrix
Roger W
RW
Duda
Robert L
RL
Steven
Alasdair C
AC
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*
D036022:Capsid Proteins; D021122:Protein Subunits; D014758:Viral Core Proteins