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A bipartite structural organization defines the SERINC family of HIV-1 restriction factors

Nature Structural & Molecular Biology volume 27, pages 78–83 (2020)Cite this article

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Abstract

The human integral membrane protein SERINC5 potently restricts HIV-1 infectivity and sensitizes the virus to antibody-mediated neutralization. Here, using cryo-EM, we determine the structures of human SERINC5 and its orthologue from Drosophila melanogaster at subnanometer and near-atomic resolution, respectively. The structures reveal a novel fold comprised of ten transmembrane helices organized into two subdomains and bisected by a long diagonal helix. A lipid binding groove and clusters of conserved residues highlight potential functional sites. A structure-based mutagenesis scan identified surface-exposed regions and the interface between the subdomains of SERINC5 as critical for HIV-1-restriction activity. The same regions are also important for viral sensitization to neutralizing antibodies, directly linking the antiviral activity of SERINC5 with remodeling of the HIV-1 envelope glycoprotein.

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Fig. 1: The structure of DmSERINC.
Fig. 2: Potential functional sites identified in SERINC structure.
Fig. 3: Structure of human SERINC5 bound to Fab.
Fig. 4: SERINC5 residues critical for HIV-1-restriction activity.

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Data availability

The cryo-EM maps and the refined atomic model of DmSERINC were deposited in the EMDB and wwPDB, respectively, with accession codes EMD-10277, EMD-10279, and PDB 6SP2. Source data for Figs. 4a, 4b, 4c, 4e and for Extended Data Figs. 1a, 1c, 1e, 1e, 2b, 3b, 4g−i, 6e−g, are available with the paper online.

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Acknowledgements

We thank the UK Biological Services Division at the National Institute of Biological Standards and Control for their expertise in animal husbandry for the production of the antibody; R. Peat and the Cell Services Platform (Crick Institute) for upscaling of the hybridoma culture and purification of the antibody; J. Diffley for advice on codon-optimization for protein expression in yeast; J. Frigola and G. Coster for the generous gift of JF1 cells and pGC014; R. Carzaniga for the maintenance of Vitrobot and Tecnai G2 microscope and user training; P. Walker, A. Purkiss, and M. Oliveira for computer and software support; M. Silva dos Santos for assistance with lipid preparations; D. Wu (University of Oxford) for lipidomics support; the UK National Institute for Biological Standards and Control, and depositor H. Katinger, for providing anti-HIV-1 4E10, 2F5, and p55/p24 antibodies; A. Engelman (Dana-Farber Cancer Institute) and J. Luban (University of Massachusetts) for comments on the manuscript. P.J.S. and R.A.C. were funded by Wellcome Trust (208361/Z/17/Z). Research in P.J.S.’s lab is supported by the MRC (MR/S009213/1) and BBSRC (BB/P01948X/1, BB/R002517/1, BB/S003339/1). This project made use of time on ARCHER and JADE granted via the UK High-End Computing Consortium for Biomolecular Simulation, HECBioSim (http://www.hecbiosim.ac.uk), supported by EPSRC (grant no. EP/R029407/1). This research was funded by US National Institutes of Health grant P50 AI150481 and the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001061), the UK Medical Research Council (FC001061), and the Wellcome Trust (FC001061).

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Author notes

  1. These author contributed equally: Annachiara Rosa, Cinzia Bertelli.

Authors and Affiliations

  1. Chromatin Structure and Mobile DNA Laboratory, Francis Crick Institute, London, UK

    Valerie E. Pye, Annachiara Rosa, Allison Ballandras-Colas & Peter Cherepanov

  2. Department of Cellular, Computational and Integrative Biology, University of Trento, Povo, Italy

    Cinzia Bertelli & Massimo Pizzato

  3. Physical and Theoretical Chemistry Laboratory, South Parks Road, Oxford, UK

    Weston B. Struwe, Idlir Liko & Carol V. Robinson

  4. Biological Mass Spectrometry and Proteomics Laboratory, MRC Laboratory of Molecular Biology, Cambridge, UK

    Sarah L. Maslen & J. Mark Skehel

  5. Department of Biochemistry, University of Oxford, Oxford, UK

    Robin Corey & Phillip J. Stansfeld

  6. National Institute for Biological Standards and Control, Hertfordshire, UK

    Mark Hassall, Giada Mattiuzzo & Yasuhiro Takeuchi

  7. Structural Biology Science Technology Platform, Francis Crick Institute, London, UK

    Andrea Nans

  8. UCL Division of Infection and Immunity, The Rayne Building, London, UK

    Yasuhiro Takeuchi

  9. School of Life Sciences & Department of Chemistry, University of Warwick, Warwick, UK

    Phillip J. Stansfeld

  10. Department of Infectious Disease, Imperial College London, St Mary’s Campus, Norfolk Place, London, UK

    Peter Cherepanov

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Contributions

V.E.P. expressed, purified, and characterized DmSERINC, built the atomic model, and developed and conducted thermostability assays; V.E.P., P.C., and A.B.-C. prepared and screened cryo-EM grids; A.N. collected all cryo-EM data; V.E.P. and P.C. refined the DmSERINC structure; A.R. and P.C. generated the stable cell line for SERINC5 expression, purified and characterized SERINC5, and determined the structure; A.R. conducted thermostability assays on SERINC5 and purified the Fab; P.C. produced mutant SERINC5 constructs; M.P. and C.B. developed and performed assays to measure surface exposure, restriction activity and virion incorporation of SERINC5 variants; W.B.S., I.L. and C.V.R. analyzed the lipid composition of DmSERINC preparations; S.L.M. and J.M.S. designed, conducted, and analyzed HDX/MS experiments; R.C. and P.J.S. conducted MD simulations on lipid-imbedded DmSERINC; M.H., G.M., and Y.T. generated hybridomas for the monoclonal anti-SERINC5 antibody; P.C. and M.P. conceived and directed the work; V.E.P., M.P., and P.C. wrote the manuscript with contributions from all authors.

Corresponding authors

Correspondence to Massimo Pizzato or Peter Cherepanov.

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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 Restriction activity and surface exposure of SERINC.

a,b, HIV-1 restriction activity of DmSERINC compared with human SERINC5 and SERINC2. Human and DmSERINC proteins with HA tags at their C termini were expressed in HEK293T cells with two different expression vectors (pcDNA and pBJ6), which provide high and low expression, respectively. Levels of the indicated SERINC proteins were assessed by Western blotting, using an anti-HA antibody (uncropped blot images are shown in the Source Data) (a) and by flow cytometry (b) to detect the proteins surface expression (b, top) or total expression (b, bottom) using an anti-FLAG antibody on non-permeabilized and permeabilized cells, respectively. c, Effect of SERINC expression on infectivity of HIV-1 produced in HEK293T cells transfected to express the indicated SERINC-iFLAG-HA and Nef-deficient HIV-1NL4-3. Data shown are mean and s.d. of n = 4 technical replicates. Data are provided in the Source Data. d, Insertion of the FLAG epitope into ECL4 does not interfere with the anti-HIV-1 restriction activity of SERINC5. Infectivity of Nef-deficient HIV-1NL4-3 produced in HEK293T cells transfected to express unmodified human SERINC5-HA or a variant modified by inserting a FLAG tag within its ECL4 (SERINC5-iFLAG-HA). Two different expression vectors (pcDNA and pBJ6) were used in order to obtain high and low SERINC5 expression, as shown above. Data shown are mean and s.d. of n = 4 technical replicates. Data are provided in the Source Data. e, Effect of ECL5 SERINC5 variants on HIV-1 susceptibility to neutralization. IC50 values derived from fitted sigmoidal curves shown in Fig. 4, obtained from quadruplicate repeats using antibodies 2F5 and 4E10 on Nef-defective HIV-1NL4-3 pseudotyped with the envelope glycoprotein derived from HIV-1JR-FL, produced by transfecting HEK293T cells with the indicated pBJ5-SERINC5-iFLAG-HA variants or the empty vector control. Data shown are mean and 95% confidence interval of n = 4 technical replicates. Data are provided in the Source Data.

Source data

Extended Data Fig. 2 Human SERINC5 purification and EM.

a, Size exclusion chromatography profile. b, Left, SDS-PAGE analysis of resulting fractions; right, cleavage of the TwinStrep tag and deglycosylation (uncropped gel images are shown in the Source Data). c, Sample micrograph of negatively stained particles. d, Representative 2D class averages. e, Schematic of image processing and reconstruction of the human SERINC5 cryo-EM structure. Details are given in Extended Methods. f, Left, Gold standard FSC curve for the cryo-EM reconstruction of SERINC5. Right, Euler angle distribution plot for particles included in the final 3D reconstruction; 3DFCS reports a sphericity of 0.976. g, The map colored according to local resolution estimated with blocres.

Source data

Extended Data Fig. 3 DmSERINC purification and EM.

a, Left, chromatography profile of DmSERINC on a Superdex 200 column; the blue arrow highlights elution of the material, which was re-injected onto the column. Right, elution profile of hexameric DmSERINC. b, Left, SDS-PAGE analysis of chromatography fractions. Right, purified hexamer (first four lanes) and monomer (last four lanes) uncleaved vs cleaved sample showing higher oligomeric states in hexamer sample shift upon cleavage of the C-terminal TwinStrep tag (uncropped gel images are shown in the Source Data). c, Sample micrograph of negatively stained DmSERINC sample from 9.8-ml peak. d, 2D class averages of negatively stained DmSERINC. e, Schematic of image processing and 3D reconstruction of the DmSERINC hexamer. Volumes are shown at two contour levels, toward the protein level in solid white and the outline of the detergent micelle in transparent gray. Details of the image processing and reconstruction are given in Extended Methods. f, Left, Gold standard FSC curve for the refined DmSERINC cryo-EM map. f, Right, Euler angle distribution plot for aligned particles contributing to the 3D reconstruction; bar lengths and color (blue, low; red, high) correspond to numbers of particles in corresponding orientations. g, Cryo-EM map colored according to local resolution estimated with blocres and shown at high (left) and low (right) contour levels. h, Cryo-EM maps of the asymmetrical DmSERINC hexamer (corresponding to 3D classes 3 and 8 in Extended Data Fig. 3e) with fitted model: viewed down six-fold axis (top) or from the side (bottom). The map is contoured to highlight the protein components (right) or the detergent micelle (left).

Source data

Extended Data Fig. 4 Structural features of DmSERINC.

a, Transmembrane topology diagram of DmSERINC structure, with residues not resolved in the cryo-EM map shaded in gray. b, Topology diagram of the SERINC protein fold, colored as in Fig. 1b. ECLs and ICLs are labeled, with disulfide bonds and subdomains A and B. c, Scatter plot of top 500 results from an analysis using Dali server, showing numbers of aligned residues versus r.m.s. deviations (Å) of Cα atom positions. d, DmSERINC hexamer colored by conservation; Guillemet indicates the viewpoint on the protomer-protomer interface labeled with an asterisk that is shown in the side view on the right. e, Examples of DmSERINC cryo-EM map with fitted model. f, Two disulfide bonds identified on the extracellular side of DmSERINC. Left, Cryo-EM map showing profile of the Cys71-Cys91 disulfide bond within ECL1. Right, Cryo-EM map showing profile of the Cys238-Cys299 disulfide bond between ECL3 and ECL4. g−i, Thermostability of the DmSERINC hexamer (g), monomer (h), and SERINC5 (i) with the addition of reducing agents (0.5 mM DTT and 0.5 mM TCEP); data shown are mean and s.d. n = 3 or 4 technical replicates. Data are provided in Source Data. j, Molecular dynamic simulations of solvation. Top left, Density analysis of waters (blue surface) around DmSERINC (gray cartoon) in one repeat of an atomistic 230-ns simulation. Bottom left, Water density shown as a 2D heat map slice. Right, DmSERINC residues implicated in controlling water wire highlighted in green.

Source data

Extended Data Fig. 5 Lipidomics of DmSERINC structure.

a, Cryo-EM map features of DmSERINC showing similarities with cardiolipin viewed with (right) and without (left) coordinates built, from two angles. b, Positions of the tentative cardiolipin sandwiched between the protomers of the hexamer. c–e, Identification of lipids associated with DmSERINC by MS. c, Lipidomics LC-MS analysis of hexameric DmSERINC5 purified from yeast cells. Ions corresponding to phospholipids (PE, PC, PI) and cardiolipin compositions are indicated. d, Structures within each lipid class were confirmed by MS/MS fragmentation. Neutral loss fragments, such as R1COO− and R2COO− ions, are diagnostic for PE, PC, PI, and cardiolipin (CL). e, Native mass spectra of DmSERINC monomers (10+ to 15+ charge state distribution) isolated from LMNG micelles spiked with PC, PG, PE, or CL lipids added at a 1:1 molar ratio. Up to two equivalents of bound CL were observed, whereas no distinct binding was detected for PC, PG, or PE.

Extended Data Fig. 6 Lipid screening.

a, Lipid binding groove apparent in DmSERINC structure. Top left, Surface representation of DmSERINC monomer revealing a groove formed between TMs 5, 7, 8, and 4. Top right, Lipid moiety modeled into the groove, shown in spheres, illustrating complementary size, shape, and location for lipid binding. Bottom left, Cartoon representation of the same view with helices labeled and colored as Fig. 1b. Bottom right, Cartoon representation with lipid shown in stick format. b, Cryo-EM map has lipid-like features in this groove. Left, map with PS modeled in. Right, Map carved to 2.5 Å around the modeled PS to highlight the lipid-like map features. c, View of DmSERINC in a POPC membrane, after 215 ns of atomistic simulation. The protein is shown as a blue cartoon and transparent surface, and the POPC lipids are red, orange, and gray spheres. Lipids in front of the protein have been removed to reveal how the protein sits in the membrane. d, View of DmSERINC after 215 ns atomistic MD simulation, showing a POPC lipid bound to the groove between TM5 and TM8. The protein is shown as a white cartoon, with the lipid in green, red, and gold spheres. Note that this lipid remains bound for the full simulation. e−g, Lipid thermostability assay. e, Change in thermostability of DmSERINC hexamer upon the addition of a specific lipid. f, Change in thermostability of DmSERINC monomer upon the addition of a specific lipid. g, Change in thermostability of SERINC5 upon the addition of a specific lipid (selected sample of lipids). Data shown in e,f are mean and s.d. of n = 3−6 technical replicates. Data are provided in Source Data.

Source data

Extended Data Fig. 7 HDX of lipid interactions with DmSERINC.

a, Left: Peptide coverage of DmSERINC monomer for HDX. Right: Structure of DmSERINC (with undefined loops modeled in using SWISS-MODEL) with coverage highlighted in blue. b–e, HDX profile of purified monomeric DmSERINC in LMNG micelles prior to (b) or after spiking with exogenous DPPS (c), sulfatides (d), or PC (e). Peptide residue numbers are shown on the x axis. f, Protected regions determined by HDX mapped onto the DmSERINC structure and highlighted in red (with undefined loops modeled in using SWISS-MODEL). g, Surface representation of DmSERINC structure colored as in Fig. 1b. with protected regions highlighted in red.

Extended Data Fig. 8 Juxtaposition of SERINC5 and the trimeric HIV-1 envelope spike.

The model of human SERINC5 is shown in gray cartoons with residues important for restriction highlighted in blue and modeled loops in transparent white. The illustrative model of full-length trimeric HIV-1 Env was assembled using PDB 6E8W (model 1; pinks) and PDB 5FUU (gp41 browns; gp120 purples). MPER (653−683) is shown in cyans. All structures are shown in cartoons; the membrane is in a cream color. a, Side-by-side comparison. b, Models shown in closer proximity. c, 90o rotation and zoom of model in b, showing that the distance between ECL5 and ECL3 is approximately the same distance (~30 Å) as that between MPER α-helices in gp41.

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2 and Supplementary Note (Extended Methods).

Reporting Summary

Supplementary Video 1

Structure of DmSERINC

Supplementary Video 2

Structure of SERINC5

Source data

Source Data Fig. 4

Statistical Source Data for Fig 4 a, b, e

Source Data Fig. 4

Unprocessed Western Blots for Fig 4c

Source Data Extended Data Fig. 1

Statistical Source Data for Extended Data fig 1 c-e

Source Data Extended Data Fig. 1

Unprocessed Western Blots

Source Data Extended Data Fig. 2

Unprocessed SDS PAGE gels

Source Data Extended Data Fig. 3

Unprocessed SDS PAGE gels

Source Data Extended Data Fig. 4

Statistical Source Data for Extended Data fig 4 g-i

Source Data Extended Data Fig. 6

Statistical Source Data for Extended Data fig 6e-g

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Pye, V.E., Rosa, A., Bertelli, C. et al. A bipartite structural organization defines the SERINC family of HIV-1 restriction factors. Nat Struct Mol Biol 27, 78–83 (2020). https://doi.org/10.1038/s41594-019-0357-0

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