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Structural and dynamical characterization of tubular HIV-1 capsid protein assemblies by solid state nuclear magnetic resonance and electron microscopy.

ABSTRACT: The wild-type HIV-1 capsid protein (CA) self-assembles in vitro into tubular structures at high ionic strength. We report solid state nuclear magnetic resonance (NMR) and electron microscopy measurements on these tubular CA assemblies, which are believed to contain a triangular lattice of hexameric CA proteins that is similar or identical to the lattice of capsids in intact HIV-1. Mass-per-length values of CA assemblies determined by dark-field transmission electron microscopy indicate a variety of structures, ranging from single-wall tubes to multiwall tubes that approximate solid rods. Two-dimensional (2D) solid state (13)C--(13)C and (15)N--(13)C NMR spectra of uniformly (15)N,(13)C-labeled CA assemblies are highly congested, as expected for a 25.6 kDa protein in which nearly the entire amino acid sequence is immobilized. Solid state NMR spectra of partially labeled CA assemblies, expressed in 1,3-(13)C(2)-glycerol medium, are better resolved, allowing the identification of individual signals with line widths below 1 ppm. Comparison of crosspeak patterns in the experimental 2D spectra with simulated patterns based on solution NMR chemical shifts of the individual N-terminal (NTD) and C-terminal (CTD) domains indicates that NTD and CTD retain their individual structures upon self-assembly of full-length CA into tubes. 2D (1)H-(13)C NMR spectra of CA assemblies recorded under solution NMR conditions show relatively few signals, primarily from segments that link the alpha-helices of NTD and CTD and from the N- and C-terminal ends. Taken together, the data support the idea that CA assemblies contain a highly ordered 2D protein lattice in which the NTD and CTD structures are retained and largely immobilized.

SUBMITTER: Chen B

PROVIDER: S-EPMC2867012 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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Structural and dynamical characterization of tubular HIV-1 capsid protein assemblies by solid state nuclear magnetic resonance and electron microscopy.

Chen Bo B Tycko Robert R

Protein science : a publication of the Protein Society 20100401 4

The wild-type HIV-1 capsid protein (CA) self-assembles in vitro into tubular structures at high ionic strength. We report solid state nuclear magnetic resonance (NMR) and electron microscopy measurements on these tubular CA assemblies, which are believed to contain a triangular lattice of hexameric CA proteins that is similar or identical to the lattice of capsids in intact HIV-1. Mass-per-length values of CA assemblies determined by dark-field transmission electron microscopy indicate a variety ...[more]

PMID: 20095046

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Project description:The HIV-1 capsid protein (CA) forms the capsid shell that encloses RNA within a mature HIV-1 virion. Previous studies by electron microscopy have shown that the capsid shell is primarily a triangular lattice of CA hexamers, with variable curvature that destroys the ideal symmetry of a planar lattice. The mature CA lattice depends on CA dimerization, which occurs through interactions between helix 9 segments of the C-terminal domain (CTD) of CA. Several high-resolution structures of the CTD-CTD dimerization interface have been reported, based on X-ray crystallography and multidimensional solution nuclear magnetic resonance (NMR), with significant differences in amino acid side chain conformations and helix 9-helix 9 orientations. In a structural model for tubular CA assemblies based on cryogenic electron microscopy (cryoEM) [Zhao et al. Nature, 2013, 497, 643-646], the dimerization interface is substantially disordered. The dimerization interface structure in noncrystalline CA assemblies and the extent to which this interface is structurally ordered within a curved lattice have therefore been unclear. Here we describe solid state NMR measurements on the dimerization interface in tubular CA assemblies, which contain the curved triangular lattice of a mature virion, including quantitative measurements of intermolecular and intramolecular distances using dipolar recoupling techniques, solid state NMR chemical shifts, and long-range side chain-side chain contacts. When combined with restraints on the distance and orientation between helix 9 segments from the cryoEM study, the solid state NMR data lead to a unique high-resolution structure for the dimerization interface in the noncrystalline lattice of CA tubes. These results demonstrate that CA lattice curvature is not dependent on disorder or variability in the dimerization interface. This work also demonstrates the feasibility of local structure determination within large noncrystalline assemblies formed by high-molecular-weight proteins, using modern solid state NMR methods.

Dataset Information

Structural and dynamical characterization of tubular HIV-1 capsid protein assemblies by solid state nuclear magnetic resonance and electron microscopy.

Publications

Structural and dynamical characterization of tubular HIV-1 capsid protein assemblies by solid state nuclear magnetic resonance and electron microscopy.

Similar Datasets

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