Manman Lu

Cyclophilin A stabilizes the HIV-1 capsid through a novel non-canonical binding site

The host cell factor cyclophilin A (CypA) interacts directly with the HIV-1 capsid and regulates viral infectivity. Although the crystal structure of CypA in complex with the N-terminal domain of the HIV-1 capsid protein (CA) has been known for nearly two decades, how CypA interacts with the viral capsid and modulates HIV-1 infectivity remains unclear. We determined the cryoEM structure of CypA in complex with the assembled HIV-1 capsid at 8-Å resolution. The structure exhibits a distinct CypA-binding pattern in which CypA selectively bridges the two CA hexamers along the direction of highest curvature. EM-guided all-atom molecular dynamics simulations and solid-state NMR further reveal that the CypA-binding pattern is achieved by single-CypA molecules simultaneously interacting with two CA subunits, in different hexamers, through a previously uncharacterized non-canonical interface. These results provide new insights into how CypA stabilizes the HIV-1 capsid and is recruited to facilitate HIV-1 infection.

Dynamic allostery governs cyclophilin A-HIV capsid interplay

Significance The mechanisms of how Cyclophilin A (CypA) regulates HIV-1 infectivity remain poorly understood. We examined the role of dynamics in capsid (CA) protein assemblies by magic-angle-spinning NMR. The assembled CA is highly dynamic. Dipolar tensors calculated from molecular dynamics trajectories are in quantitative agreement with the NMR results. Motions in the CypA loop are sequence-dependent and attenuated in the escape mutants A92E and G94D. Dynamics are similar in escape mutants and CA/CypA complex. These findings suggest that CA escapes from CypA dependence through dynamic allostery. Thus, a host factors function in HIV infectivity may not be primarily associated with a structural change of the capsid core, but with altering its dynamics, such as the reduction of motions for the CypA loop. Host factor protein Cyclophilin A (CypA) regulates HIV-1 viral infectivity through direct interactions with the viral capsid, by an unknown mechanism. CypA can either promote or inhibit viral infection, depending on host cell type and HIV-1 capsid (CA) protein sequence. We have examined the role of conformational dynamics on the nanosecond to millisecond timescale in HIV-1 CA assemblies in the escape from CypA dependence, by magic-angle spinning (MAS) NMR and molecular dynamics (MD). Through the analysis of backbone 1H-15N and 1H-13C dipolar tensors and peak intensities from 3D MAS NMR spectra of wild-type and the A92E and G94D CypA escape mutants, we demonstrate that assembled CA is dynamic, particularly in loop regions. The CypA loop in assembled wild-type CA from two strains exhibits unprecedented mobility on the nanosecond to microsecond timescales, and the experimental NMR dipolar order parameters are in quantitative agreement with those calculated from MD trajectories. Remarkably, the CypA loop dynamics of wild-type CA HXB2 assembly is significantly attenuated upon CypA binding, and the dynamics profiles of the A92E and G94D CypA escape mutants closely resemble that of wild-type CA assembly in complex with CypA. These results suggest that CypA loop dynamics is a determining factor in HIV-1s escape from CypA dependence.

CryoEM Structure Refinement by Integrating NMR Chemical Shifts with Molecular Dynamics Simulations.

Single particle cryoEM has emerged as a powerful method for structure determination of proteins and complexes, complementing X-ray crystallography and NMR spectroscopy. Yet, for many systems, the resolution of cryoEM density map has been limited to 4-6 Å, which only allows for resolving bulky amino acids side chains, thus hindering accurate model building from the density map. On the other hand, experimental chemical shifts (CS) from solution and solid state MAS NMR spectra provide atomic level data for each amino acid within a molecule or a complex; however, structure determination of large complexes and assemblies based on NMR data alone remains challenging. Here, we present a novel integrated strategy to combine the highly complementary experimental data from cryoEM and NMR computationally by molecular dynamics simulations to derive an atomistic model, which is not attainable by either approach alone. We use the HIV-1 capsid protein (CA) C-terminal domain as well as the large capsid assembly to demonstrate the feasibility of this approach, termed NMR CS-biased cryoEM structure refinement.

Dynamic Nuclear Polarization enhanced MAS NMR spectroscopy for structural analysis of HIV-1 protein assemblies

Mature infectious HIV-1 virions contain conical capsids composed of CA protein, generated by the proteolytic cleavage cascade of the Gag polyprotein, termed maturation. The mechanism of capsid core formation through the maturation process remains poorly understood. We present DNP-enhanced MAS NMR studies of tubular assemblies of CA and Gag CA-SP1 maturation intermediate and report 20-64-fold sensitivity enhancements due to DNP at 14.1 T. These sensitivity enhancements enabled direct observation of spacer peptide 1 (SP1) resonances in CA-SP1 by dipolar-based correlation experiments, unequivocally indicating that the SP1 peptide is unstructured in assembled CA-SP1 at cryogenic temperatures, corroborating our earlier results. Furthermore, the dependence of DNP enhancements and spectral resolution on magnetic field strength (9.4-18.8 T) and temperature (109-180 K) was investigated. Our results suggest that DNP-based measurements could potentially provide residue-specific dynamics information by allowing for the extraction of the temperature dependence of the anisotropic tensorial or relaxation parameters. With DNP, we were able to detect multiple well-resolved isoleucine side-chain conformers; unique intermolecular correlations across two CA molecules; and functionally relevant conformationally disordered states such as the 14-residue SP1 peptide, none of which are visible at ambient temperatures. The detection of isolated conformers and intermolecular correlations can provide crucial constraints for structure determination of these assemblies. Overall, our results establish DNP-based MAS NMR spectroscopy as an excellent tool for the characterization of HIV-1 assemblies.

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

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 time scales. Chemical shift anisotropy (CSA) tensors, recorded in magic-angle-spinning NMR experiments, provide direct residue-specific probes of motions on nano- to microsecond time scales. We combined NMR, MD, and density-functional-theory calculations, to gain quantitative understanding of internal backbone dynamics in CA assemblies, and we 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.

Magic angle spinning NMR of viruses

Viruses, relatively simple pathogens, are able to replicate in many living organisms and to adapt to various environments. Conventional atomic-resolution structural biology techniques, X-ray crystallography and solution NMR spectroscopy provided abundant information on the structures of individual proteins and nucleic acids comprising viruses; however, viral assemblies are not amenable to analysis by these techniques because of their large size, insolubility, and inherent lack of long-range order. In this article, we review the recent advances in magic angle spinning NMR spectroscopy that enabled atomic-resolution analysis of structure and dynamics of large viral systems and give examples of several exciting case studies.

MAS NMR of HIV-1 protein assemblies

The negative global impact of the AIDS pandemic is well known. In this perspective article, the utility of magic angle spinning (MAS) NMR spectroscopy to answer pressing questions related to the structure and dynamics of HIV-1 protein assemblies is examined. In recent years, MAS NMR has undergone major technological developments enabling studies of large viral assemblies. We discuss some of these evolving methods and technologies and provide a perspective on the current state of MAS NMR as applied to the investigations into structure and dynamics of HIV-1 assemblies of CA capsid protein and of Gag maturation intermediates.

19F Magic Angle Spinning NMR Spectroscopy and Density Functional Theory Calculations of Fluorosubstituted Tryptophans: Integrating Experiment and Theory for Accurate Determination of Chemical Shift Tensors

The 19F chemical shift is a sensitive NMR probe of structure and electronic environment in organic and biological molecules. In this report, we examine chemical shift parameters of 4F-, 5F-, 6F-, and 7F-substituted crystalline tryptophan by magic angle spinning (MAS) solid-state NMR spectroscopy and density functional theory. Significant narrowing of the 19F lines was observed under fast MAS conditions, at spinning frequencies above 50 kHz. The parameters characterizing the 19F chemical shift tensor are sensitive to the position of the fluorine in the aromatic ring and, to a lesser extent, the chirality of the molecule. Accurate calculations of 19F magnetic shielding tensors require the PBE0 functional with a 50% admixture of a Hartree-Fock exchange term, as well as taking account of the local crystal symmetry. The methodology developed will be beneficial for 19F-based MAS NMR structural analysis of proteins and protein assemblies.

Chemical Shifts of the Carbohydrate Binding Domain of Galectin-3 from Magic Angle Spinning NMR and Hybrid Quantum Mechanics/Molecular Mechanics Calculations

Magic angle spinning NMR spectroscopy is uniquely suited to probe the structure and dynamics of insoluble proteins and protein assemblies at atomic resolution, with NMR chemical shifts containing rich information about biomolecular structure. Access to this information, however, is problematic, since accurate quantum mechanical calculation of chemical shifts in proteins remains challenging, particularly for 15NH. Here we report on isotropic chemical shift predictions for the carbohydrate recognition domain of microcrystalline galectin-3, obtained from using hybrid quantum mechanics/molecular mechanics (QM/MM) calculations, implemented using an automated fragmentation approach, and using very high resolution (0.86 Å lactose-bound and 1.25 Å apo form) X-ray crystal structures. The resolution of the X-ray crystal structure used as an input into the AF-NMR program did not affect the accuracy of the chemical shift calculations to any significant extent. Excellent agreement between experimental and computed shifts is obtained for 13Cα, while larger scatter is observed for 15NH chemical shifts, which are influenced to a greater extent by electrostatic interactions, hydrogen bonding, and solvation.