Experimental Characterization of the Hepatitis B Virus Capsid Dynamics by Solid-State NMR

Abstract

Protein plasticity and dynamics are important aspects of their function. Here we use solid-state NMR to experimentally characterize the dynamics of the 3.5 MDa hepatitis B virus (HBV) capsid, assembled from 240 copies of the Cp149 core protein. We measure both T1 and T1ρ relaxation times, which we use to establish detectors on the nanosecond and microsecond timescale. We compare our results to those from a 1 microsecond all-atom Molecular Dynamics (MD) simulation trajectory for the capsid. We show that, for the constituent residues, nanosecond dynamics are faithfully captured by the MD simulation. The calculated values can be used in good approximation for the NMR-non-detected residues, as well as to extrapolate into the range between the nanosecond and microsecond dynamics, where NMR has a blind spot at the current state of technology. Slower motions on the microsecond timescale are difficult to characterize by all-atom MD simulations owing to computational expense, but are readily accessed by NMR. The two methods are, thus, complementary, and a combination thereof can reliably characterize motions covering correlation times up to a few microseconds.

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

“…We have recently shown that the WGCF system supports phosphorylation of viral proteins 19 , 67 , 68 , and were therefore able to analyze the phosphorylation state of CF-Cp183 in the presence of CAMs. The 31 P spectrum of CF-Cp183 in absence of CAM indicates that no phosphorylation occurred in these capsids (since no 31 P signal at 6 ppm is observed), while clear signals for Cp-associated RNA were seen around −1.5 ppm (Fig.…”

“…All spectra were referenced using DSS and recorded at a sample temperature of 4 °C in the 3.2 mm probe and between 20 and 25 °C in the 1.3 mm probe, as determined by the resonance frequency of the supernatant water 85 . For dynamics measurements, site-specific solid-state NMR relaxation-rate constants R 1ρ ( 15 N) were measured on 2 H 13 C 15 N Cp149 alone and in interaction with JNJ-890, using the 3D hCANH sequence described in reference 19 in an 0.7 mm rotor at 80 kHz MAS, 20 T (850 MHz) external magnetic field strength and with a 13 kHz spin-lock field. Eight 3D hCANH experiments were recorded with the spin-lock delay time varied between 1 µs and 251 ms. For further experimental information see Supplementary Table 5 .…”

“…Typically, the Cp spikes have recently been described to be involved in nuclear export of the encapsidated viral RNA, with the tip containing a sensor for secretion 15 , 16 . While the flexibility of the spike tips has been repeatedly reported 17 – 19 , its functional importance is not firmly settled, but might be important in adequately presenting the nuclear export signals, and also other envelopment-relevant motifs. Also, the capsid carries a continuous flow of rcDNA into the nucleus via the core protein’s nuclear localization signals located in the CTD.…”

Rotating Frame Relaxation in Magic Angle Spinning Solid State NMR, a Promising Tool for Characterizing Biopolymer Motion

“…One popular format for solid state R 1ρ measurements of biopolymers involves amidic 1 H detected experiments which is an aspect of solid state NMR that has had many advances recently . Commonly, these experiments involve recording the relaxation of the attached 15 N, for which angular fluctuations of the amide functional group would be expected to modulate both the anisotropic chemical shift of the amidic 15 N and the dipolar coupling to the directly attached proton. − Some studies additionally probe the relaxation of the detected 1 H. ,, In general, these studies are carried out at relatively high applied magnetic field strength (>700 MHz), use fast magic-angle spinning (ν r ≥ 50 kHz), and often also involve at least partial deuteration of the protein sample. With these conditions, protons can be detected with excellent sensitivity.…”

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

“…We have recently shown that the WGCF system supports phosphorylation of viral proteins 61–63 , and were therefore able to analyze the phosphorylation state of CF-Cp183 in the presence of CAMs. The 31 P spectrum of CF-Cp183 in absence of CAM indicates that no phosphorylation occurred in these capsids (since no 31 P signal at 6 ppm is observed), while clear signals for Cp-associated RNA were seen around −1.5 ppm ( Figure 4b ) (the RNA packaged by the capsid during cell-free synthesis is the mRNA coding for the protein 59 ).…”