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Structural basis of CD4 downregulation by HIV-1 Nef

Nature Structural & Molecular Biology volume 27, pages 822–828 (2020)Cite this article

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Abstract

The HIV-1 Nef protein suppresses multiple immune surveillance mechanisms to promote viral pathogenesis and is an attractive target for the development of novel therapeutics. A key function of Nef is to remove the CD4 receptor from the cell surface by hijacking clathrin- and adaptor protein complex 2 (AP2)-dependent endocytosis. However, exactly how Nef does this has been elusive. Here, we describe the underlying mechanism as revealed by a 3.0-Å crystal structure of a fusion protein comprising Nef and the cytoplasmic domain of CD4 bound to the tetrameric AP2 complex. An intricate combination of conformational changes occurs in both Nef and AP2 to enable CD4 binding and downregulation. A pocket on Nef previously identified as crucial for recruiting class I MHC is also responsible for recruiting CD4, revealing a potential approach to inhibit two of Nef’s activities and sensitize the virus to immune clearance.

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Fig. 1: In vitro assembly and crystal structure of the Nef, CD4 cytoplasmic domain and clathrin AP2 complex.
Fig. 2: Recruitment of CD4 cytoplasmic domain and the role of Nef N-terminal loop.
Fig. 3: Nef binding induces conformational change in the β2 subunit of AP2.
Fig. 4: The downregulations of MHC-I and CD4 are distinct both mechanistically and structurally, yet their cytoplasmic domains share a common binding site on Nef.

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Data availability

The coordinates and structure factors for the crystal structure have been deposited at the Protein Data Bank (PDB) with the accession code 6URI. The proteomics XL-MS data have been deposited at the ProteomeXchange database69 with the accession code PXD019338. The integrative structural model has been deposited at PDB-Dev with the accession code PDBDEV_00000050. Source data are provided with this paper.

Code availability

Files containing the input data, scripts and output results for the integrative structure modeling of the Nef-CD4CD–AP2Δμ2-CTD complex are available at https://github.com/integrativemodeling/Nef_CD4_AP2.

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Acknowledgements

We thank Y. Xiong (Yale University) for helpful discussions and valuable input. We thank the beamline staff at the Advanced Photon Source beamline 24-ID and the National Synchrotron Light Source beamline 17-ID. We thank J. Bonifacino (National Institutes of Health (NIH)) for providing the gene of rat α adaptin. This work was supported by the University of Massachusetts Dartmouth startup fund (X.J.) and US NIH grants no. AI102778 and no. AI129706 (J.G.). R.M.K., I.E., A.S. and N.K. were supported by NIH grant no. P50AI150476. R.M.K. was also supported by NIH fellowship grant no. F32AI127291. A.S. was also supported by NIH grants no. U19AI135990, no. R01GM083960, no. P41GM109824 and no. S10OD021596. N.K. was also supported by NIH grants no. P50GM082250 and no. U19AI135990.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, Dartmouth, MA, USA

    Yonghwa Kwon, Mohammad Karimian Shamsabadi, Jacob Kress & Xiaofei Jia

  2. Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA

    Robyn M. Kaake & Nevan Krogan

  3. Gladstone Institutes, San Francisco, CA, USA

    Robyn M. Kaake & Nevan Krogan

  4. Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, USA

    Ignacia Echeverria & Andrej Sali

  5. The VA San Diego Healthcare System, San Diego, CA, USA

    Marissa Suarez, Charlotte Stoneham, Rajendra Singh & John Guatelli

  6. Department of Medicine, University of California, San Diego, La Jolla, CA, USA

    Charlotte Stoneham, Peter W. Ramirez, Rajendra Singh & John Guatelli

  7. Department of Pharmaceutical Chemistry and Quantitative Biosciences Institute, University of California, San Francisco, San Francisco, CA, USA

    Andrej Sali

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Contributions

Y.K. performed protein expression, purification, binding assays and crystallization. Y.K. and X.J. performed data collection, structure determination, model building and refinement. M.S., C.S. and P.W.R. performed CD4 and MHC-I downregulation assays and mutagenesis. R.M.K. performed XL-MS. I.E. performed integrative modeling. Y.K. and M.K.S. performed in vitro mutagenesis and fluorescence polarization assays. J.K. and R.S. contributed to protein expression and purification. Y.K., R.M.K., I.E., A.S., N.K., J.G. and X.J. designed the experiments. All contributed to data analysis. J.G. and X.J. supervised the project. Y.K., J.G. and X.J. wrote the manuscript.

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Correspondence to Xiaofei Jia.

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Extended data

Extended Data Fig. 1 Electron density map for the N-terminal loop of Nef.

2Fo-Fc map (1σ level with B factor sharpened by −50 Å2) for Nef residues 34–40 and 47-75 is shown as black mesh. Nef residues 41-46 could not be built due to the lack of density. Density for Nef 34-40 is less defined, although sidechain density for Leu37 is clear.

Extended Data Fig. 2 β2 subunit, if intact, would clash with the bound Nef.

Overlay of the intact β2 subunit (dark red, PDB 2XA7) with β2 in the current structure (green) indicates that clashing would take place between Nef and N-terminus of the intact β2, specifically residues Asn10, Lys11, Lys12, and Gly13 (spheres).

Extended Data Fig. 3 Crosslinking mass spectrometry and integrative structure modeling of the Nef-CD4CD-AP2Δμ2-CTD complex.

a, Overview of the DSSO XL-MS3 analysis method. b, CX-Circos linkage map of all Nef-CD4CD-AP2Δμ2-CTD interlinks. c, Integrative structure of the Nef-CD4CD-AP2Δμ2-CTD complex. The localization probability density of the ensemble of structures is shown with representative (centroid) structure from the ensemble embedded within it. Regions present in the crystal structure are shown as ribbons and segments not present in the crystal structure are shown as beads. d, Histogram showing the distribution of the cross-linked Cα–Cα distances in the integrative structure. The structural ensemble satisfies 89% of the XLs used to compute it. e, RMSD between rigid-bodies in the model ensemble. The vertical axis corresponds to the rigid body used as reference for superimposition and the horizontal axis are the rigid bodies for which the average RMSD was computed. f, Detail of crosslinks mapped to Nef. Satisfied and violated crosslinks shown in green and pink, respectively. g, Positioning of the unfolded β2 segment.

Extended Data Fig. 4 Binding of Nef N-terminal helix to the Nef core is incompatible with CD4 downregulation.

N-terminal helix of Nef (8-23) is modeled into the current structure. Red dotted line represents the would-be distance between residues 23 and 34, which cannot be covered by ten residues (Nef 24-33).

Extended Data Fig. 5 Nef residues at the CD4 -binding pocket are highly conserved.

Nef sequences from HIV sequence compendium 2017 were analyzed though multiple sequence alignment (HIV sequence database, www.hiv.lanl.gov). Alignment was done in HXB2 convention (bottom) and important residues are additionally labeled using the NL4.3 convention on top. D123, shown in red text, is important for both CD4 and MHC-I downregulation. Other residues important for CD4 downregulation are in cyan and black texts. Black texts refer to residues, in addition to D123, that surround CD4. Other residues important for MHC-I downregulation are in orange. The logo representation, with the height of each letter proportional to the observed frequency of the corresponding amino acid residue, was generated by WebLogo70.

Extended Data Fig. 6 The unique conformation of Nef N-terminal loop observed in the current structure is incompatible with Nef dimerization.

Nef in current conformation (cyan, cartoon) is overlaid with the SH2-SH3-dependent Nef dimer39 (dark blue and red envelopes, PDB 4U5W). While majority of Nef in the current structure overlays well with the Nef protomer shown as the dark blue envelope, the N-terminal region of Nef (circled) intrudes severely into the volume of the other Nef protomer (red envelope).

Supplementary information

Supplementary Information

Supplementary Fig. 1, containing the uncropped gels for Figs. 2h and 4b,c.

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Supplementary Table 1

Intra- and inter-subunit DSSO inter-linked residues of Nef-CD4CD-AP2Δμ2-CTD.

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Source Data Fig. 2

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Kwon, Y., Kaake, R.M., Echeverria, I. et al. Structural basis of CD4 downregulation by HIV-1 Nef. Nat Struct Mol Biol 27, 822–828 (2020). https://doi.org/10.1038/s41594-020-0463-z

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