Abstract
HK97 is a double-stranded DNA bacteriophage that undergoes dramatic conformational changes during viral capsid maturation and for which x-ray structures, at near atomic resolution, of multiple intermediate and mature capsid states are available. Both amide H/(2)H exchange and crystallographic comparisons between the pre-expanded Prohead II particles and the expanded Head II of bacteriophage HK97 revealed quaternary interactions that remain fixed throughout maturation and appear to maintain intercapsomer integrity at all quasi- and icosahedral 3-fold axes. These 3-fold staples are formed from Arg and Glu residues and a metal binding site. Mutations of either Arg-347 or Arg-194 or a double mutation of E344Q and E363A resulted in purification of the phage in capsomer form (hexamers and pentamers). Mutants that did assemble had both decreased thermal stability and decreased in vitro expansion rates. Amide H/(2)H exchange mass spectrometry showed that in the wild type capsid some subunits had a bent "spine" helix (highly exchanging), whereas others were straight (less exchanging). Similar analysis of the never assembled mutant capsomers showed uniform amide exchange in all of these that was higher than that of the straight spine helices (characterized in more mature intermediates), suggesting that the spine helix is somewhat bent prior to capsid assembly. The result further supports a previously proposed mechanism for capsid expansion in which the delta domains of each subunit induce a high energy intermediate conformation, which now appears to include a bent helix during capsomer assembly.
Highlights
HK97 is a double-stranded DNA bacteriophage that undergoes dramatic conformational changes during viral capsid maturation and for which x-ray structures, at near atomic resolution, of multiple intermediate and mature capsid states are available
We recently showed with amide H/2H exchange and crystallographic comparisons between the pre-expanded P-II particles and the mature Head II (H-II) that maturation is probably guided by tertiary structure twisting and secondary structure changes around a fixed set of intercapsomer interactions that surround all quasi- and icosahedral 3-fold axes in the capsid shell [12]
Mutating either of the glutamate residues individually did not prevent assembly, mutating both did as seen in the E344Q/E363A mutant
Summary
P-I, Prohead I; DSC, differential scanning calorimetry; P-II, Prohead II; H-I, Head I; H-II, Head II; WT, wild type; Q/A, E344Q/E363A; EI, expansion intermediate; EM, electron microscopy. Shown to undergo a large scale twisting motion, causing hinging in all three P-domain -strands (see Fig. 8A for domain nomenclature) These data imply that interactions at the 3-fold interface may be crucial in assembling the capsid from individual capsomers as well as providing a fixed point from which subunits bend while maintaining intercapsomer contacts. We confirmed this role for the 3-fold interactions by mutagenesis of relevant residues and characterized the resulting assembly products, thermal stabilities, and maturation kinetics. Amide exchange of the spine helix in the mutant capsomers was compared with previously characterized particle forms as well as P-I and WT capsomers disassembled from P-I
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