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Atomic-resolution structure of HIV-1 capsid tubes by magic-angle spinning NMR

Nature Structural & Molecular Biology volume 27, pages 863–869 (2020)Cite this article

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Abstract

HIV-1 capsid plays multiple key roles in viral replication, and inhibition of capsid assembly is an attractive target for therapeutic intervention. Here, we report the atomic-resolution structure of capsid protein (CA) tubes, determined by magic-angle spinning NMR and data-guided molecular dynamics simulations. Functionally important regions, including the NTD β-hairpin, the cyclophilin A-binding loop, residues in the hexamer central pore, and the NTD-CTD linker region, are well defined. The structure of individual CA chains, their arrangement in the pseudo-hexameric units of the tube and the inter-hexamer interfaces are consistent with those in intact capsids and substantially different from the organization in crystal structures, which feature flat hexamers. The inherent curvature in the CA tubes is controlled by conformational variability of residues in the linker region and of dimer and trimer interfaces. The present structure reveals atomic-level detail in capsid architecture and provides important guidance for the design of novel capsid inhibitors.

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Fig. 1: MAS-NMR spectra and structure of the hexameric unit in CA tubular assemblies.
Fig. 2: Structure of an HIV-1 CA tube generated by data-guided molecular dynamics.
Fig. 3: Difference between the MAS-NMR and X-ray structures of the hexameric unit in CA tubular assemblies.

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Data availability

The coordinates of the atomic structures have been deposited in the Protein Data Bank under accession codes PDB 6WAP for the single CA chain and PDB 6X63 for the CA tube. MAS-NMR chemical shifts, dihedral constraints and distance constraints have been deposited in the Biological Magnetic Resonance Data Bank under accession code 30741.

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Acknowledgements

This work was supported by the National Institutes of Health (NIH; grant no. P50AI1504817). We acknowledge the support of the National Science Foundation (NSF; grant no. CHE0959496) for acquisition of the 850-MHz NMR spectrometer, the NIH (grant no. P30GM110758) for support for the core instrumentation infrastructure at the University of Delaware and also grant no. S10OD012213 for acquisition of the 750-MHz NMR spectrometer at the University of Pittsburgh. This work was partially supported by the Intramural Research Program of the Center for Information Technology at the NIH. This research used resources from the Oak Ridge Leadership Computing Facility (OLCF) at Oak Ridge National Laboratory, which is supported by the Office of Science of the Department of Energy under contract no. DE-AC05-00OR22725. We acknowledge a Director’s Discretionary Award on the Summit supercomputer from the OLCF.

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  1. Guangjin Hou

    Present address: State Key Laboratory of Catalysis, Dalian Institute of Chemical Physics, The Chinese Academy of Sciences, Dalian, P. R. China

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Delaware, Newark, DE, USA

    Manman Lu, Ryan W. Russell, Alexander J. Bryer, Caitlin M. Quinn, Guangjin Hou, Huilan Zhang, Juan R. Perilla & Tatyana Polenova

  2. Pittsburgh Center for HIV Protein Interactions, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

    Manman Lu, Ryan W. Russell, Alexander J. Bryer, Juan R. Perilla, Angela M. Gronenborn & Tatyana Polenova

  3. Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

    Manman Lu & Angela M. Gronenborn

  4. Imaging Sciences Laboratory, Center for Information Technology, National Institutes of Health, Bethesda, MD, USA

    Charles D. Schwieters

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Contributions

T.P. and A.M.G. conceived the project and guided the work. J.R.P. designed and guided the MD simulations and structure calculations of the CA tube. M.L. prepared the samples, performed NMR experiments and analyzed the experimental data. R.W.R. and M.L. performed the structure calculations of the CA hexamer unit. C.M.Q. assisted in the structure calculations of the hexamer unit. A.J.B. conducted the MD simulations and structure calculation of the CA tube. M.L., R.W.R. and A.J.B. prepared figures for the manuscript. R.W.R. and A.J.B. wrote scripts for analysis of calculation results and visualization of the hexamer unit and tube, respectively. C.D.S. provided critical input in the NIH-Xplor-based structure calculations. C.M.Q., G.H. and H.Z. took part in the design or analysis of NMR experiments. T.P. and A.M.G. took the lead in writing the manuscript. All authors discussed the results and contributed to manuscript preparation.

Corresponding authors

Correspondence to Juan R. Perilla, Angela M. Gronenborn or Tatyana Polenova.

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The authors declare no competing interests.

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Peer review information Inês Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Summary of MAS-derived NMR distances.

a, Inter-residue contact plot. b, Intrachain distances (gray dotted lines), mapped onto the ribbon diagram structure of a single CA chain. NTD helices are colored purple, the β-hairpin yellow, loops gray, and the CTD helices cyan.

Extended Data Fig. 2 Initial MAS NMR structure of full-length CA protein.

Ensemble of 10 lowest-energy structures for the single-chain. Best-fit superpositions for the NTD (a) and CTD (b), respectively, are shown. The color coding is identical to that in Extended Data Fig. 1.

Extended Data Fig. 3 Protocol for the structure determination of a CA hexamer using MAS NMR and cryo-EM data.

Ensembles of the 10 lowest-energy structures at each step are depicted in ribbon representation, with NTD helices colored purple, the β-hairpin yellow, loops gray, and CTD helices cyan.

Extended Data Fig. 4 Structural clusters of HIV-1 CA tube.

a–f, Plots of k-medoid clusters4 for different structural units in the final tube structure by Silhouette analysis5. g–l, Conformations from clustering analyses and the number of members within each cluster. g, Representative structures (medoids) of dimer interface clusters. h, Alignment of the two trimer interface medoids yielded by clustering. i, CypA loops grouped and colored by conformational cluster. j, Two structural clusters from analysis of monomeric β-hairpins. k, Nine clusters from analysis of hexameric β-hairpins. l, Flexible linkers grouped into four conformational clusters.

Extended Data Fig. 5 Water occupancy in the MD simulation of the HIV-1 CA tube.

a, Water occupancy map, averaged over 80 ns of the trajectory. For each frame in the trajectory, a binary mask is used to indicate the presence of water at every point in space. The average of these masks for a trajectory is the water occupancy map, and each point in the latter describes the fractional occupancy of water at a specific location. 3-D visualizations, isosurfaces, are generated according to the value at each point in the occupancy map and are shown at varying occupancy cutoffs. b, Histogram of dimer interfaces involved in water-mediated contacts, the latter determined by proximity of S149, E175 and W184 side chain atoms within 4.0 Å of the water occupancy map at the specified occupancy cutoff values. c, Illustration of a dimer interface with the side chains of S149, E175 and W184 in stick representation and an isosurface of the water occupancy map (red; > 0.70 fractional occupancy).

Extended Data Fig. 6 Analysis of trimer interfaces in the HIV-1 CA tube.

a, A hexamer of hexamers, highlighting the two trimer interfaces: one involving chains A, D and F (blue) and the other between chains B, C and E (orange), shown from two perspectives. b, Histogram of the shortest L205 Cα–Cα distance at each trimer interface in the CA tube, for the orange and blue interfaces. The histogram is drawn as a frequency polygon, where vertices represent the center of each bin along the x-axis. c, Best-fit superposition of the two medoids from clustering analysis of the trimer interface (blue, red) and the cryo-electron tomography (cryo-ET) structure (accession codes: EMDB 3475, PDB ID 5MD7)26.

Extended Data Fig. 7 Molecular dynamics simulation setup for the HIV-1 CA tube.

a, The 13.9-million atom system comprises water molecules (transparent), Na+ and Cl- ions (yellow and blue, respectively), and protein (white). View of the dimer (b) and trimer (c) interface docked into the 8.6 Å cryo-EM envelope4 (accession code EMD-5582). d, Fourier Shell Correlation (FSC) between the 8.6 Å CA tube density and simulated 8.6 Å densities of the CA tube before (blue) and after (red) data-guided MD refinement. The dashed lines in the plot correspond to FSC = 0.50 and FSC = 0.143, for resolutions of 10.0 Å and 8.3 Å, respectively. e, Scaling analysis of the MD simulations of the current system on the Oak Ridge National Laboratory’s Summit supercomputer.

Extended Data Fig. 8 Superposition of synthetic peak positions, back calculated from the MAS NMR structure of a hexamer unit and the CORD spectrum of [1,6-13C]-glucose,U-15N-CA tubular assemblies.

The spectrum was collected at 14.1 T, with a MAS frequency of 14 kHz, and a CORD mixing time of 500 ms. Cross peak positions were calculated for the lowest-energy structure of the hexameric unit, using the experimental 13C chemical shifts, and all intrachain and interchain 13C-13C contacts, corresponding to distances up to 7 Å. Peak positions corresponding to intrachain contacts are colored red and blue for distances up to 5 Å and those between 5 Å and 7 Å, respectively. Peak positions corresponding to interchain contacts are colored in green. Peak positions due to isotope scrambling are colored yellow.

Extended Data Fig. 9 Superposition of synthetic peak positions, back calculated from the MAS NMR structure of a hexamer unit, and the CORD spectrum of U-13C,15N-CA tubular assemblies.

The spectrum was collected at 14.1 T, a MAS frequency of 14 kHz, and a CORD mixing time of 500 ms. Cross peak positions were calculated for the lowest-energy structure of the hexameric unit, using the experimental 13C chemical shifts, and all intrachain and interchain 13C-13C contacts corresponding to distances up to 7 Å. Peak positions corresponding to intrachain contacts are colored red and blue for distances up to 5 Å and those between 5 Å and 7 Å, respectively. Peak positions corresponding to interchain contacts are colored in green.

Extended Data Fig. 10 Interchain contacts (up to 7 Å) identified in the CORD spectra extracted from simulated cross peak positions.

Interchain correlations are mapped on two neighboring CA chains in the lowest-energy structure of the hexameric unit. Intermolecular correlations are shown as green dashed lines and the associated residues are depicted in orange stick representation.

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Lu, M., Russell, R.W., Bryer, A.J. et al. Atomic-resolution structure of HIV-1 capsid tubes by magic-angle spinning NMR. Nat Struct Mol Biol 27, 863–869 (2020). https://doi.org/10.1038/s41594-020-0489-2

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