TY - JOUR
T1 - The integrity of the intradimer interface of the hepatitis B virus capsid protein dimer regulates capsid self-assembly
AU - Hadden-Perilla, Jodi A.
AU - Zlotnick, Adam
AU - Zhao, Zhongchao
AU - Wang, Joseph Che Yen
AU - Segura, Carolina Perez
N1 - Funding Information:
This work was supported by NIH grant R01-AI144022 to A.Z. and NSF grant MCB-2027096, funded in part by Delaware EPSCoR, to J.A.H.-P. Some electron microscopy was supported by a grant from the Indiana CTSI to A.Z. We gratefully acknowledge the use of the Indiana University Physical Biochemistry Instrumentation Facility and the Indiana University Electron Microscopy Center. Molecular dynamics components of this research are part of the Blue Waters sustained-petascale computing project, which is supported by the National Science Foundation (awards OCI-0725070 and ACI-1238993) and the state of Illinois. Blue Waters is a joint effort of the University of Illinois at Urbana−Champaign and its National Center for Supercomputing Applications. The authors acknowledge the use of Blue Waters through allocation awards to J.A.H.-P.
Funding Information:
This work was supported by NIH grant R01-AI144022 to A.Z. and NSF grant MCB-2027096, funded in part by Delaware EPSCoR, to J.A.H.-P. Some electron microscopy was supported by a grant from the Indiana CTSI to A.Z. We gratefully acknowledge the use of the Indiana University Physical Biochemistry Instrumentation Facility and the Indiana University Electron Microscopy Center. Molecular dynamics components of this research are part of the Blue Waters sustained-petascale computing project, which is supported by the National Science Foundation (awards OCI-0725070 and ACI-1238993) and the state of Illinois. Blue Waters is a joint effort of the University of Illinois at Urbana–Champaign and its National Center for Supercomputing Applications. The authors acknowledge the use of Blue Waters through allocation awards to J.A.H.-P.
Publisher Copyright:
© 2020 American Chemical Society.
PY - 2020/12/18
Y1 - 2020/12/18
N2 - During the hepatitis B virus lifecycle, 120 copies of homodimeric capsid protein assemble around a copy of reverse transcriptase and viral RNA and go on to produce an infectious virion. Assembly needs to be tightly regulated by protein conformational change to ensure symmetry, fidelity, and reproducibility. Here, we show that structures at the intradimer interface regulate conformational changes at the distal interdimer interface and so regulate assembly. A pair of interacting charged residues, D78 from each monomer, conspicuously located at the top of a four-helix bundle that forms the intradimer interface, were mutated to serine to disrupt communication between the two monomers. The mutation slowed assembly and destabilized the dimer to thermal and chemical denaturation. Mutant dimers showed evidence of transient partial unfolding based on the appearance of new proteolytically sensitive sites. Though the mutant dimer was less stable, the resulting capsids were as stable as the wildtype, based on assembly and thermal denaturation studies. Cryo-EM image reconstructions of capsid indicated that the subunits adopted an “open” state more usually associated with a free dimer and that the spike tips were either disordered or highly flexible. Molecular dynamics simulations provide mechanistic explanations for these results, suggesting that D78 stabilizes helix 4a, which forms part of the intradimer interface, by capping its N-terminus and hydrogen-bonding to nearby residues, whereas the D78S mutation disrupts these interactions, leading to partial unwinding of helix 4a. This in turn weakens the connection from helix 4 and the intradimer interface to helix 5, which forms the interdimer interface.
AB - During the hepatitis B virus lifecycle, 120 copies of homodimeric capsid protein assemble around a copy of reverse transcriptase and viral RNA and go on to produce an infectious virion. Assembly needs to be tightly regulated by protein conformational change to ensure symmetry, fidelity, and reproducibility. Here, we show that structures at the intradimer interface regulate conformational changes at the distal interdimer interface and so regulate assembly. A pair of interacting charged residues, D78 from each monomer, conspicuously located at the top of a four-helix bundle that forms the intradimer interface, were mutated to serine to disrupt communication between the two monomers. The mutation slowed assembly and destabilized the dimer to thermal and chemical denaturation. Mutant dimers showed evidence of transient partial unfolding based on the appearance of new proteolytically sensitive sites. Though the mutant dimer was less stable, the resulting capsids were as stable as the wildtype, based on assembly and thermal denaturation studies. Cryo-EM image reconstructions of capsid indicated that the subunits adopted an “open” state more usually associated with a free dimer and that the spike tips were either disordered or highly flexible. Molecular dynamics simulations provide mechanistic explanations for these results, suggesting that D78 stabilizes helix 4a, which forms part of the intradimer interface, by capping its N-terminus and hydrogen-bonding to nearby residues, whereas the D78S mutation disrupts these interactions, leading to partial unwinding of helix 4a. This in turn weakens the connection from helix 4 and the intradimer interface to helix 5, which forms the interdimer interface.
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U2 - 10.1021/acschembio.0c00277
DO - 10.1021/acschembio.0c00277
M3 - Article
C2 - 32459465
AN - SCOPUS:85090912110
VL - 15
SP - 3124
EP - 3132
JO - ACS Chemical Biology
JF - ACS Chemical Biology
SN - 1554-8929
IS - 12
ER -