Solid-State NMR Studies of HIV-1 Capsid Protein Assemblies

Han, Yun; Ahn, Jin-Woo; Concel, Jason; Byeon, In‐Ja L.; Gronenborn, Angela M.; Yang, Jun

doi:10.1021/ja908687k

Cited by 109 publications

(170 citation statements)

References 78 publications

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“…CA (HXB2, NL4-3, NL4-3/A92E, and NL4-3/G94D variants) and CypA were expressed and purified by using the same protocol as reported (14,41). Tubular assemblies of CA protein were prepared and their morphology characterized as described (14) and detailed in SI Appendix, SI Text.…”

Section: Methodsmentioning

confidence: 99%

Dynamic allostery governs cyclophilin A–HIV capsid interplay

Hou

Zhang

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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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-1's escape from CypA dependence.

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Section: Methodsmentioning

confidence: 99%

Dynamic allostery governs cyclophilin A–HIV capsid interplay

Hou

Zhang

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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153

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“…The conical assemblies of U- 13 C, 15 N-Tyr-labeled C-terminal domain (CTD) of HIV-1 CA protein were prepared by controlled precipitation from 50% w/v solution of PEG-4000 at the ratio of 1:1 (v/v) with the final protein concentration of 16 mg/mL, as reported previously. 38 15 N]-Ala were packed into 3.2 mm rotors, and all spectra were recorded at MAS frequencies of 10 and 20 kHz, controlled to within ±0.005 kHz. The n.a.…”

Section: Introductionmentioning

confidence: 99%

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

Hou

Byeon

Ahn

et al. 2012

The Journal of Chemical Physics

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C and15 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

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“…Although capsids within mature HIV-1 often have an approximately conical morphology, large morphological variations have been reported (10), as well as variations in apparent closure (11). Studies of pure CA performed in vitro have also found a wide variety of morphologies for CA assemblies, including tubes with 40 -60 nm diameters (3,4,(12)(13)(14)(15), spheres with minimum diameters of 20 nm and maximum diameters exceeding 10 m (2, 13, 16), planar sheets (2), and assemblies with asymmetric morphologies resembling those within mature HIV-1 (7,13,16).…”

mentioning

confidence: 99%

Major Variations in HIV-1 Capsid Assembly Morphologies Involve Minor Variations in Molecular Structures of Structurally Ordered Protein Segments

Lü¹,

Bayro

Tycko

2016

Journal of Biological Chemistry

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We present the results of solid state nuclear magnetic resonance (NMR) experiments on HIV-1 capsid protein (CA) assemblies with three different morphologies, namely wild-type CA (WT-CA) tubes with 35-60 nm diameters, planar sheets formed by the Arg 18 -Leu mutant (R18L-CA), and R18L-CA spheres with 20 -100 nm diameters. The experiments are intended to elucidate molecular structural variations that underlie these variations in CA assembly morphology. We find that multidimensional solid state NMR spectra of 15 N, 13 C-labeled CA assemblies are remarkably similar for the three morphologies, with only small differences in 15 N and 13 C chemical shifts, no significant differences in NMR line widths, and few differences in the number of detectable NMR cross-peaks. Thus, the pronounced differences in morphology do not involve major differences in the conformations and identities of structurally ordered protein segments. Instead, morphological variations are attributable to variations in conformational distributions within disordered segments, which do not contribute to the solid state NMR spectra. Variations in solid state NMR signals from certain amino acid side chains are also observed, suggesting differences in the intermolecular dimerization interface between curved and planar CA lattices, as well as possible differences in intramolecular helix-helix packing.

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Solid-State NMR Studies of HIV-1 Capsid Protein Assemblies

Cited by 109 publications

References 78 publications

Dynamic allostery governs cyclophilin A–HIV capsid interplay

Dynamic allostery governs cyclophilin A–HIV capsid interplay

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

Major Variations in HIV-1 Capsid Assembly Morphologies Involve Minor Variations in Molecular Structures of Structurally Ordered Protein Segments

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