Guangjin Hou

Motions on the millisecond time scale and multiple conformations of HIV-1 capsid protein: implications for structural polymorphism of CA assemblies.

The capsid protein (CA) of human immunodeficiency virus 1 (HIV-1) assembles into a cone-like structure that encloses the viral RNA genome. Interestingly, significant heterogeneity in shape and organization of capsids can be observed in mature HIV-1 virions. In vitro, CA also exhibits structural polymorphism and can assemble into various morphologies, such as cones, tubes, and spheres. Many intermolecular contacts that are critical for CA assembly are formed by its C-terminal domain (CTD), a dimerization domain, which was found to adopt different orientations in several X-ray and NMR structures of the CTD dimer and full-length CA proteins. Tyr145 (Y145), residue two in our CTD construct used for NMR structure determination, but not present in the crystallographic constructs, was found to be crucial for infectivity and engaged in numerous interactions at the CTD dimer interface. Here we investigate the origin of CA structural plasticity using solid-state NMR and solution NMR spectroscopy. In the solid state, the hinge region connecting the NTD and CTD is flexible on the millisecond time scale, as evidenced by the backbone motions of Y145 in CA conical assemblies and in two CTD constructs (137-231 and 142-231), allowing the protein to access multiple conformations essential for pleimorphic capsid assemblies. In solution, the CTD dimer exists as two major conformers, whose relative populations differ for the different CTD constructs. In the longer CTD (144-231) construct that contains the hinge region between the NTD and CTD, the populations of the two conformers are likely determined by the protonation state of the E175 side chain that is located at the dimer interface and within hydrogen-bonding distance of the W184 side chain on the other monomer. At pH 6.5, the major conformer exhibits the same dimer interface as full-length CA. In the short CTD (150-231) construct, no pH-dependent conformational shift is observed. These findings suggest that the presence of structural plasticity at the CTD dimer interface permits pleiotropic HIV-1 capsid assembly, resulting in varied capsid morphologies.

1H-13C/1H-15N heteronuclear dipolar recoupling by R-symmetry sequences under fast magic angle spinning for dynamics analysis of biological and organic solids.

Fast magic angle spinning (MAS) NMR spectroscopy is becoming increasingly important in structural and dynamics studies of biological systems and inorganic materials. Superior spectral resolution due to the efficient averaging of the dipolar couplings can be attained at MAS frequencies of 40 kHz and higher with appropriate decoupling techniques, while proton detection gives rise to significant sensitivity gains, therefore making fast MAS conditions advantageous across the board compared with the conventional slow- and moderate-MAS approaches. At the same time, many of the dipolar recoupling approaches that currently constitute the basis for structural and dynamics studies of solid materials and that are designed for MAS frequencies of 20 kHz and below, fail above 30 kHz. In this report, we present an approach for (1)H-(13)C/(1)H-(15)N heteronuclear dipolar recoupling under fast MAS conditions using R-type symmetry sequences, which is suitable even for fully protonated systems. A series of rotor-synchronized R-type symmetry pulse schemes are explored for the determination of structure and dynamics in biological and organic systems. The investigations of the performance of the various RN(n)(v)-symmetry sequences at the MAS frequency of 40 kHz experimentally and by numerical simulations on [U-(13)C,(15)N]-alanine and [U-(13)C,(15)N]-N-acetyl-valine, revealed excellent performance for sequences with high symmetry number ratio (N/2n > 2.5). Further applications of this approach are presented for two proteins, sparsely (13)C/uniformly (15)N-enriched CAP-Gly domain of dynactin and U-(13)C,(15)N-Tyr enriched C-terminal domain of HIV-1 CA protein. Two-dimensional (2D) and 3D R16(3)(2)-based DIPSHIFT experiments carried out at the MAS frequency of 40 kHz, yielded site-specific (1)H-(13)C/(1)H-(15)N heteronuclear dipolar coupling constants for CAP-Gly and CTD CA, reporting on the dynamic behavior of these proteins on time scales of nano- to microseconds. The R-symmetry-based dipolar recoupling under fast MAS is expected to find numerous applications in studies of protein assemblies and organic solids by MAS NMR spectroscopy.

Probing structure and dynamics of protein assemblies by magic angle spinning NMR spectroscopy.

In living organisms, biological molecules often organize into multicomponent complexes. Such assemblies consist of various proteins and carry out essential functions, ranging from cell division, transport, and energy transduction to catalysis, signaling, and viral infectivity. To understand the biological functions of these assemblies, in both healthy and disease states, researchers need to study their three-dimensional architecture and molecular dynamics. To date, the large size, the lack of inherent long-range order, and insolubility have made atomic resolution studies of many protein assemblies challenging or impractical using traditional structural biology methods such as X-ray diffraction and solution NMR spectroscopy. In the past 10 years, we have focused our work on the development and application of magic angle spinning solid-state NMR (MAS NMR) methods to characterize large protein assemblies at atomic-level resolution. In this Account, we discuss the rapid progress in the field of MAS NMR spectroscopy, citing work from our laboratory and others on methodological developments that have facilitated the in-depth analysis of biologically important protein assemblies. We emphasize techniques that yield enhanced sensitivity and resolution, such as fast MAS (spinning frequencies of 40 kHz and above) and nonuniform sampling protocols for data acquisition and processing. We also discuss the experiments for gaining distance restraints and for recoupling anisotropic tensorial interactions under fast MAS conditions. We give an overview of sample preparation approaches when working with protein assemblies. Following the overview of contemporary MAS NMR methods, we present case studies into the structure and dynamics of two classes of biological systems under investigation in our laboratory. We will first turn our attention to cytoskeletal microtubule motor proteins including mammalian dynactin and dynein light chain 8. We will then discuss protein assemblies from the HIV-1 retrovirus.

Enhanced Sensitivity by Nonuniform Sampling Enables Multidimensional MAS NMR Spectroscopy of Protein Assemblies

We report dramatic sensitivity enhancements in multidimensional MAS NMR spectra by the use of nonuniform sampling (NUS) and introduce maximum entropy interpolation (MINT) processing that assures the linearity between the time and frequency domains of the NUS acquired data sets. A systematic analysis of sensitivity and resolution in 2D and 3D NUS spectra reveals that with NUS, at least 1.5- to 2-fold sensitivity enhancement can be attained in each indirect dimension without compromising the spectral resolution. These enhancements are similar to or higher than those attained by the newest-generation commercial cryogenic probes. We explore the benefits of this NUS/MaxEnt approach in proteins and protein assemblies using 1-73-(U-(13)C,(15)N)/74-108-(U-(15)N) Escherichia coli thioredoxin reassembly. We demonstrate that in thioredoxin reassembly, NUS permits acquisition of high-quality 3D-NCACX spectra, which are inaccessible with conventional sampling due to prohibitively long experiment times. Of critical importance, issues that hinder NUS-based SNR enhancement in 3D-NMR of liquids are mitigated in the study of solid samples in which theoretical enhancements on the order of 3-4 fold are accessible by compounding the NUS-based SNR enhancement of each indirect dimension. NUS/MINT is anticipated to be widely applicable and advantageous for multidimensional heteronuclear MAS NMR spectroscopy of proteins, protein assemblies, and other biological systems.

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.

Multidimensional magic angle spinning NMR spectroscopy for site-resolved measurement of proton chemical shift anisotropy in biological solids.

The proton chemical shift (CS) tensor is a sensitive probe of structure and hydrogen bonding. Highly accurate quantum-chemical protocols exist for computation of (1)H magnetic shieldings in the various contexts, making proton chemical shifts potentially a powerful predictor of structural and electronic properties. However, (1)H CS tensors are not yet widely used in protein structure calculation due to scarcity of experimental data. While isotropic proton shifts can be readily measured in proteins even in the solid state, determination of the (1)H chemical shift anisotropy (CSA) tensors remains challenging, particularly in molecules containing multiple proton sites. We present a method for site-resolved measurement of amide proton CSAs in fully protonated solids under magic angle spinning. The approach consists of three concomitant 3D experiments yielding spectra determined by either mainly (1)H CSA, mainly (1)H–(15)N dipolar, or combined (1)H CSA and (1)H–(15)N dipolar interactions. The anisotropic interactions are recoupled using RN-sequences of appropriate symmetry, such as R12(1)(4), and (15)N/(13)C isotropic CS dimensions are introduced via a short selective (1)H–(15)N cross-polarization step. Accurate (1)H chemical shift tensor parameters are extracted by simultaneous fit of the lineshapes recorded in the three spectra. An application of this method is presented for an 89-residue protein, U-(13)C,(15)N-CAP-Gly domain of dynactin. The CSA parameters determined from the triple fits correlate with the hydrogen-bonding distances, and the trends are in excellent agreement with the prior solution NMR results. This approach is generally suited for recording proton CSA parameters in various biological and organic systems, including protein assemblies and nucleic acids.

Magic Angle Spinning NMR Reveals Sequence-Dependent Structural Plasticity, Dynamics, and the Spacer Peptide 1 Conformation in HIV-1 Capsid Protein Assemblies

A key stage in HIV-1 maturation toward an infectious virion requires sequential proteolytic cleavage of the Gag polyprotein leading to the formation of a conical capsid core that encloses the viral RNA genome and a small complement of proteins. The final step of this process involves severing the SP1 peptide from the CA-SP1 maturation intermediate, which triggers the condensation of the CA protein into the capsid shell. The details of the overall mechanism, including the conformation of the SP1 peptide in CA-SP1, are still under intense debate. In this report, we examine tubular assemblies of CA and the CA-SP1 maturation intermediate using magic angle spinning (MAS) NMR spectroscopy. At magnetic fields of 19.9 T and above, outstanding quality 2D and 3D MAS NMR spectra were obtained for tubular CA and CA-SP1 assemblies, permitting resonance assignments for subsequent detailed structural characterization. Dipolar- and scalar-based correlation experiments unequivocally indicate that SP1 peptide is in a random coil conformation and mobile in the assembled CA-SP1. Analysis of two CA protein sequence variants reveals that, unexpectedly, the conformations of the SP1 tail, the functionally important CypA loop, and the loop preceding helix 8 are modulated by residue variations at distal sites. These findings provide support for the role of SP1 as a trigger of the disassembly of the immature CA capsid for its subsequent de novo reassembly into mature cores and establish the importance of sequence-dependent conformational plasticity in CA assembly.

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.

Recoupling of chemical shift anisotropy by R-symmetry sequences in magic angle spinning NMR spectroscopy.

(13)C and (15)N chemical shift (CS) interaction is a sensitive probe of structure and dynamics in a wide variety of biological and inorganic systems, and in the recent years several magic angle spinning NMR approaches have emerged for residue-specific measurements of chemical shift anisotropy (CSA) tensors in uniformly and sparsely enriched proteins. All of the currently existing methods are applicable to slow and moderate magic angle spinning (MAS) regime, i.e., MAS frequencies below 20 kHz. With the advent of fast and ultrafast MAS probes capable of spinning frequencies of 40-100 kHz, and with the superior resolution and sensitivity attained at such high frequencies, development of CSA recoupling techniques working under such conditions is necessary. In this work, we present a family of R-symmetry based pulse sequences for recoupling of (13)C∕(15)N CSA interactions that work well in both natural abundance and isotopically enriched systems. We demonstrate that efficient recoupling of either first-rank (σ(1)) or second-rank (σ(2)) spatial components of CSA interaction is attained with appropriately chosen γ-encoded RN(n)(v) symmetry sequences. The advantage of these γ-encoded RN(n)(v)-symmetry based CSA (RNCSA) recoupling schemes is that they are suitable for CSA recoupling under a wide range of MAS frequencies, including fast MAS regime. Comprehensive analysis of the recoupling properties of these RN(n)(v) symmetry sequences reveals that the σ(1)-CSA recoupling symmetry sequences exhibit large scaling factors; however, the partial homonuclear dipolar Hamiltonian components are symmetry allowed, which makes this family of sequences suitable for CSA measurements in systems with weak homonuclear dipolar interactions. On the other hand, the γ-encoded symmetry sequences for σ(2)-CSA recoupling have smaller scaling factors but they efficiently suppress the homonuclear dipole-dipole interactions. Therefore, the latter family of sequences is applicable for measurements of CSA parameters in systems with strong homonuclear dipolar couplings, such as uniformly-(13)C labeled biological solids. We demonstrate RNCSA NMR experiments and numerical simulations establishing the utility of this approach to the measurements of (13)C and (15)N CSA parameters in model compounds, [(15)N]-N-acetyl-valine (NAV), [U-(13)C, (15)N]-alanine, [U-(13)C,(15)N]-histidine, and present the application of this approach to [U-(13)C∕(15)N]-Tyr labeled C-terminal domain of HIV-1 CA protein.

Spin Diffusion Driven by R-Symmetry Sequences: Applications to Homonuclear Correlation Spectroscopy in MAS NMR of Biological and Organic Solids

We present a family of homonuclear (13)C-(13)C magic angle spinning spin diffusion experiments, based on R2(n)(v) (n = 1 and 2, v = 1 and 2) symmetry sequences. These experiments are well suited for (13)C-(13)C correlation spectroscopy in biological and organic systems and are especially advantageous at very fast MAS conditions, where conventional PDSD and DARR experiments fail. At very fast MAS frequencies the R2(1)(1), R2(2)(1), and R2(2)(2) sequences result in excellent quality correlation spectra both in model compounds and in proteins. Under these conditions, individual R2(n)(v) display different polarization transfer efficiency dependencies on isotropic chemical shift differences: R2(2)(1) recouples efficiently both small and large chemical shift differences (in proteins these correspond to aliphatic-to-aliphatic and carbonyl-to-aliphatic correlations, respectively), while R2(1)(1) and R2(2)(2) exhibit the maximum recoupling efficiency for the aliphatic-to-aliphatic or carbonyl-to-aliphatic correlations, respectively. At moderate MAS frequencies (10-20 kHz), all R2(n)(v) sequences introduced in this work display similar transfer efficiencies, and their performance is very similar to that of PDSD and DARR. Polarization transfer dynamics and chemical shift dependencies of these R2-driven spin diffusion (RDSD) schemes are experimentally evaluated and investigated by numerical simulations for [U-(13)C,(15)N]-alanine and the [U-(13)C,(15)N] N-formyl-Met-Leu-Phe (MLF) tripeptide. Further applications of this approach are illustrated for several proteins: spherical assemblies of HIV-1 U-(13)C,(15)N CA protein, U-(13)C,(15)N-enriched dynein light chain DLC8, and sparsely (13)C/uniformly (15)N enriched CAP-Gly domain of dynactin. Due to the excellent performance and ease of implementation, the presented R2(n)(v) symmetry sequences are expected to be of wide applicability in studies of proteins and protein assemblies as well as other organic solids by MAS NMR spectroscopy.