Fast Magic‐Angle‐Spinning NMR Reveals the Evasive Hepatitis B Virus Capsid C‐Terminal Domain**

Abstract

Experimentally determined protein structures often feature missing domains. One example is the Cterminal domain (CTD) of the hepatitis B virus capsid protein, a functionally central part of this assembly, crucial in regulating nucleic-acid interactions, cellular trafficking, nuclear import, particle assembly and maturation. However, its structure remained elusive to all current techniques, including NMR. Here we show that the recently developed proton-detected fast magicangle-spinning solid-state NMR at > 100 kHz MAS allows one to detect this domain and unveil its structural and dynamic behavior. We describe the experimental framework used and compare the domain's behavior in different capsid states. The developed approaches extend solid-state NMR observations to residues characterized by large-amplitude motion on the microsecond timescale, and shall allow one to shed light on other flexible protein domains still lacking their structural and dynamic characterization.

Molecular elucidation of drug-induced abnormal assemblies of the hepatitis B virus capsid protein by solid-state NMR

“…As alternative intermolecular interactions involving the CTDs the positively charged arginine side chains can form salt-bridges with the phosphorylated sidechains from other P7-Cp183 dimers, building a densely woven interaction network which maintains the apparently globular particle shape in a similar way as when interacting with packaged RNA. This view is meanwhile experimentally supported by recent NMR measurements which show that the CTDs of P7-CP183 show a similar dynamic behavior to those of pgRNA-filled Cp183, and must thus be involved in interactions, likely of intermolecular nature, between arginine side chains and phosphorylated Ser/Thr residues 63 .…”

“…CTD-less Cp variants can be packaged RNA 60 – 62 , as its multivalent electrostatic interactions with the positively charged CTDs could also counteract particle disruption. Similarly, interactions between phosphorylated CTDs and positively charged arginine residues in P7-Cp183 can stabilize capsids 63 . Still, how DTT could impact these interactions remains to be determined.…”

Biophysics-Guided Lead Discovery of HBV Capsid Assembly Modifiers

“…2 The Nterminal domain of Cp (Cp149) is sufficient for forming regular capsids, 15 while the arginine-rich C-terminal region (residues 150−183) is needed for pregenomic RNA (pgRNA) encapsidation among other functions. 16,17 The Cp149 dimer consists of two domains: the dimerization interface consisting of helices α3 and α4 (Figure 1A) and the assembly interface responsible for forming interdimer contacts (helices α1, α2, and α5, Figure 1B). 18,19 Previous studies proposed that the Cp149 dimers trigger capsid assembly by adopting an energetically unfavorable "assembly active" conformation, which in turn leads to assembly nucleation.…”

“…A promising orthogonal approach for eliminating the infection, perhaps in combination with antiviral or immune therapies, is to target the HBV nucleocapsid assembly. − The HBV capsid comprises 240 copies of the core protein (Cp) forming an icosahedral protein shell, while the Cp primarily exists as a homodimer in solution under nonassembling (low ionic strength) conditions . The N-terminal domain of Cp (Cp149) is sufficient for forming regular capsids, while the arginine-rich C-terminal region (residues 150–183) is needed for pregenomic RNA (pgRNA) encapsidation among other functions. , The Cp149 dimer consists of two domains: the dimerization interface consisting of helices α3 and α4 (Figure A) and the assembly interface responsible for forming interdimer contacts (helices α1, α2, and α5, Figure B). , …”