HIV-1 Capsid Function Is Regulated by Dynamics: Quantitative Atomic-Resolution Insights by Integrating Magic-Angle-Spinning NMR, QM/MM, and MD

Abstract

HIV-1 CA capsid protein possesses intrinsic conformational flexibility, which is essential for its assembly into conical capsids and interactions with host factors. CA is dynamic in the assembled capsid, and residues in functionally important regions of the protein undergo motions spanning many decades of timescales. Chemical shift anisotropy (CSA) tensors, recorded in magic-angle-spinning NMR experiments, provide direct residue-specific probes of motions on nano- to microsecond timescales. We combined NMR, MD, and Density-Functional-Theory calculations, to gain quantitative understanding of internal backbone dynamics in CA assemblies, and found that the dynamically averaged 15N CSA tensors calculated by this joined protocol are in remarkable agreement with experiment. Thus, quantitative atomic-level understanding of the relationships between CSA tensors, local backbone structure and motions in CA assemblies is achieved, demonstrating the power of integrating NMR experimental data and theory for characterizing atomic-resolution dynamics in biological systems.

Expanding the horizons for structural analysis of fully protonated protein assemblies by NMR spectroscopy at MAS frequencies above 100 kHz

“…The structure of CA tubes has been characterized by various methods [41], illuminating details of the capsid architecture and its relationship to viral function. Our team has extensively studied structure and dynamics of these assemblies by heteronuclear- based MAS NMR spectroscopy at frequencies of 10–20 kHz [31; 42; 43; 44]. The samples employed possessed high conformational homogeneity and yielded excellent-resolution spectra, but, alas, at these spinning speeds 1 H chemical shifts have not been accessible.…”

“…These are the N- and C- terminal residues (1-20 and 217-231) the CypA binding loop (85-95), and the NTD-CTD flexible linker region (residues 142-149). These residues exhibit motions spanning timescales slower than nanoseconds and as slow as milliseconds [42; 44; 46], and the absence of the corresponding resonances in the spectra is not surprising. We note that these peaks are present in the 13 C-detected experiments conducted at MAS frequencies of 10–20 kHz, because these were performed at temperatures of 4–15 °C, while the lowest temperature attainable in the current study with the 0.7 mm probe was 26 °C.…”

“…HIV-1 capsids are highly dynamic entities [41; 42; 44; 46; 47], with motions occurring over many orders of magnitudes (nano to milliseconds and slower). Functionally, dynamic processes play important roles in capsid’s assembly, uncoating, and interactions with host factors [44]. We have recently interrogated nano- to microsecond timescale dynamics in HIV-1 CA assemblies using 1 H- 15 N dipolar tensors [42].…”

“…RN-symmetry based experiments and their improved versions developed by our group for accurate measurements of heteronuclear dipolar and 1 H CSA tensors at MAS frequencies of 40 kHz and below have been applied to organic and biological systems, including proteins and protein assemblies [27; 28; 29; 34; 42; 48]. In these reports, we have presented in-depth analysis of the performance of the RN-symmetry based experiments and validated their applications in protein assemblies, including the HIV-1 CA assemblies, using various means, such as empirical correlations between 1 H CSA and hydrogen bond length using electrostatic models [29], quantum chemical (DFT) calculations of CSA tensors [49], as well as a combined MD/DFT approach [44]. Others have demonstrated that 1 H CSA tensors can be recorded in small molecules at the MAS frequency of 65 kHz [50], albeit no validation was reported on the measured CSAs from quantum chemical calculations.…”

“…Indeed, 1 H CSA tensors, are another sensitive probe of dynamics, which also report on hydrogen bonding interactions [29; 49]. To extract dynamics information from CSA tensors, quantum mechanical Density Functional Theory (DFT) calculations as well as combined MD/DFT calculations can be employed, as we have demonstrated recently [44]. …”