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.

Structural basis for nuclear import of hepatitis B virus (HBV) nucleocapsid core

“…CTD compaction upon phosphorylation is achieved by an intramolecular network of side-chain contacts between phosphate moieties and CTD arginine side chains that latches the CTD polypeptide chains at distinct points, favoring a reduced spatial footprint while maintaining the accessibility of the NLS in the majority of conformers. Our data agree with a recent solid-state nuclear magnetic resonance study, which found that CTD does not form specific secondary structures in the context of RNA binding or upon phosphorylation and that phosphorylated CTDs exhibit restricted motion ( 58 ). Furthermore, the decrease in conformational space sampled by the CTD upon phosphorylation observed in MD simulations is consistent with enhanced structural stability due to the introduction of salt bridges.…”

“…The way importin α1/β1 recognizes the arginine-rich NLS is somewhat unexpected. Located at the C-terminal end of the intrinsically disordered CTD ( 58 ), the NLS could, in principle, extend as much as ~85 Å above the capsid surface. Instead, our cryo-EM reconstruction of the EC:Imp α1/β1 complex showed that importin α1 preferentially collapses onto the quasi-sixfold pore, projecting its concave surface toward the NLS that folds backward to bind importin α1 major NLS-binding site in an antiparallel manner.…”

Selective Detection of Intermediate-Amplitude Motion by Solid-State NMR

“…The intermediate-amplitude motion of cell wall polysaccharides observed here has similarities to the motion of the C-terminal domain of the hepatitis B virus capsid core protein . At 4 °C, signals of the C-terminal residues are missing in both CP-hNH spectra and INEPT-hCH spectra measured under 100 kHz MAS.…”

“…7−10 In virus capsid proteins, intrinsically disordered domains coexist with rigid domains to carry out multiple functions. 11 To understand the molecular basis for the properties of these materials and the protein aggregation process in diseases, it is important to characterize both the structure of the rigid components and the dynamics of the mobile components.…”

“…In extracellular matrices of plants, fungi, and other organisms, rigid biopolymers such as cellulose and chitin coexist with highly mobile polymers such as homogalacturonan (HG) and α-1,3-glucan − (Figure ). This coexistence imparts both mechanical strength and flexibility to the cell wall, thus protecting the plant cells while allowing them to grow. , In neurodegenerative diseases, intrinsically disordered proteins aggregate to form a rigid core that is comprised of a small portion of the protein, whereas the rest of the protein forms a disordered fuzzy coat. − In virus capsid proteins, intrinsically disordered domains coexist with rigid domains to carry out multiple functions . To understand the molecular basis for the properties of these materials and the protein aggregation process in diseases, it is important to characterize both the structure of the rigid components and the dynamics of the mobile components.…”