Cryo-EM Structure of Chikungunya Virus in Complex with the Mxra8 Receptor

Abstract

Mxra8 is a receptor for multiple arthritogenic alphaviruses that cause debilitating acute and chronic musculoskeletal disease in humans. Herein, we present a 2.2 Å resolution X-ray crystal structure of Mxra8 and 4 to 5 Å resolution cryo-electron microscopy reconstructions of Mxra8 bound to chikungunya (CHIKV) virus-like particles and infectious virus. The Mxra8 ectodomain contains two strand-swapped Ig-like domains oriented in a unique disulfide-linked head-to-head arrangement. Mxra8 binds by wedging into a cleft created by two adjacent CHIKV E2-E1 heterodimers in one trimeric spike and engaging a neighboring spike. Two binding modes are observed with the fully mature VLP, with one Mxra8 binding with unique contacts. Only the high-affinity binding mode was observed in the complex with infectious CHIKV, as viral maturation and E3 occupancy appear to influence receptor binding-site usage. Our studies provide insight into how Mxra8 binds CHIKV and creates a path for developing alphavirus entry inhibitors.

Keywords: HDX mass spectrometry; alphavirus; cryo-electron microscopy; infection; protein crystallography; surface plasmon resonance; virus receptor.

Figure 1.. Mxra8 crystal structure and topology diagram.

A. Ribbon model of the mouse Mxra8 protein structure. The β-strands are labeled following standard convention. The two Ig-like domains are colored from the N- to C-terminus in a rainbow spectrum of blue to red with the disulfides shown as yellow ball and stick bonds. The positions of the cysteines forming the interdomain disulfide bond are indicated. The N- and C-termini are labeled in lowercase. B. Secondary structure diagram of Mxra8, numbered by sequence positions in the X-ray structure. Colored arrows indicate the β-strands as in panel A. Yellow lines connect the cysteine positions forming disulfide bonds. The AB strand swaps anchor the connections between the D1 and D2 domains. C. Cartoon schematic of Mxra8, with labeled D1 and D2 domains, N- and C-termini, BC linkers, and cysteine residues forming the interdomain disulfide bond. Mxra8 is bowed. A rotation of ~155˚ around the hinge region is required for alignment of the two domains. See Fig S1, S2, S3, and Table S1.

Figure 2.. Two-dimensional cisTEM analysis of CHIKV particles with or without Mxra8 bound.

A. Raw electron micrographs of CHIKV VLPs. B. Two-dimensional classification scheme for binning of CHIKV VLPs. C-D. Two-dimensional equatorial slices of CHIKV VLP alone (C) or CHIKV VLP in complex with Mxra8 (D). Dimensions: the outer radius of the nucleocapsid shell (~180 Å), lipid bilayer (~240 Å), E1 protein glycoprotein shell (~280 Å), E2 protein spike (~340 Å), and bound Mxra8 (~350 Å) from the viral center. E-F. Fourier shell correlation (FSC) plot versus resolution for CHIKV VLP alone (E) or CHIKV VLP with bound Mxra8 (F). See Fig S1 and Table S2.

Figure 3.. Cryo-EM reconstruction of CHIKV particles with or without Mxra8 binding.

A-C. Paired, colored surface representations (top panel) and equatorial cross-sections (bottom panel) of CHIKV VLP (A), CHIKV VLP + Mxra8 (B), and local resolution of CHIKV VLP + Mxra8 (C). The white triangle indicates one icosahedral asymmetric unit. The 5-fold (i5), 3-fold (i3), and 2-fold (i2) icosahedral axes of symmetry are indicated with a pentagon, triangles, and oval, respectively. Trimeric spikes are labeled “i3” if coincident with the i3 axes, and “q3” if on a quasi 3-fold axes. Black arrows: directions of icosahedral symmetry axes (i2, i3, q3, and i5). (A-B) Radial distance color scheme: red, electron dense core and RNA; yellow, capsid; green, membrane lipid; dark blue, E1; cyan, E2 spike; and magenta, Mxra8. D. Zoomed-in view of the q3 spike, highlighting the three E2-E1 heterodimers that interact with Mxra8 at site 3: the wrapped (light grey), the intraspike (medium grey), and the interspike (dark grey) heterodimers. The i5 and i2 axes of symmetry are labeled with a pentagon and oval, respectively. Mxra8 is colored by domain (D2, dark magenta; D1, light magenta). E-F. Side views of Mxra8 at site 3. (E) Mxra8 and the wrapped, intraspike, and interspike heterodimers are colored to match panel D. (F) Mxra8 and the wrapped, intraspike, and interspike heterodimers are colored by local resolution, as reported in panel C: red, 4 Å; yellow, 6 Å; and green, 8 Å; See Table S2 and S3.

Figure 4.. Atomic model of Mxra8 interaction with CHIKV.

A. The refined model of Mxra8 and CHIKV structural proteins (E1, E2, transmembrane (TM) helices, and Capsid) in the electron density map of the CHIKV VLP with Mxra8 reconstruction at threshold σ=1, viewed from the side (left panel) and top (right panel) of the asymmetric unit. B. Cartoon of the asymmetric unit viewed from the side (left panel) and top (right panel) with all domains labeled. “q3” and “i3” refer to the icosahedral and quasi 3-fold axes, respectively. C. Individual E2-E1 subunits at the binding interface, specifically site 1 Mxra8 and its wrapped heterodimer (left panel), site 1 Mxra8 with its intraspike heterodimer (middle panel), and site 3 Mxra8 with its interspike heterodimer (right panel). Mxra8 and structural proteins are colored by domain. Mxra8: dark magenta, D2; light magenta, D1. E1: DI, light grey; DII, medium grey; DIII, dark grey; fusion loop, orange; TM region, black. E2: A domain, light cyan; β-linker, medium cyan; domain B, dark cyan; domain C, medium blue; TM region, dark blue. Capsid, forest green. Domain labels are boxed if the domain is at the binding interface. Disulfide bonds are shown as yellow balls and sticks. D. Ribbon model of the refined site 1 Mxra8 in its electron density map, with the N- to C-terminus in a rainbow spectrum of blue to red and the disulfide bonds shown as yellow balls and sticks. The N- and C-termini are labeled in lowercase, and the β-strands are labeled in uppercase. The density map is viewed at contour level =1.7. E. Zoomed-in view of the protrusion density and atomic structure of the N-linked glycan at residue 118, viewed at contour level =1.4. The value is the standard deviation of density values above the mean in the map. See Fig S2, S3, S6and Table S4.

Figure 5.. Binding competition, epitope mapping, and mutagenesis supporting the Mxra8-CHIKV cryo-EM model.

A. Competition BLI assay traces. CHIKV VLP was captured on the biosensor with an anti-CHIKV mAb and then dipped into wells containing Mxra8 ectodomain alone or with an anti-Mxra8 mAb or isotype control mAb. One experiment of three is shown. B. Surface and ribbon diagrams of Mxra8 at site 3 and the E2-E1 domains at its binding interface. Structural proteins are colored by domain. E1: DI, light grey; DII, medium grey; fusion loop, orange. E2: A domain, light cyan; β-linker, medium cyan; domain B, dark cyan. ‘ denotes domains within the intraspike heterodimer, and “ for the interspike heterodimer; the wrapped heterodimer is labelled without symbols. Surface representation of Mxra8 models are colored with the HDX-mapped epitopes of anti-Mxra8 mAbs, where shades of violet correspond to mAb 1G11.E6, green for mAbs 1H1.F5 and 3G2.F5, and yellow for mAbs 4E7.D10 and 8F7.E1. C. Trans-complementation of 293T cells (which lack endogenous MXRA8) with charge-reversal mutated Mxra8. (Top) CHIKV infection was determined flow cytometric analysis of intracellular E2 protein. (Bottom) Relative surface expression of Mxra8 by flow cytometry using anti-Mxra8 mAbs. Data are pooled from three experiments performed in triplicate, normalized to the WT controls, and the mean values are shown (one-way ANOVA with Dunnett’s post-test: *, P < 0.05; ****, P < 0.0001). D. Ribbon diagram of Mxra8 model at site 1 with side chains of mutated residues shown as balls and sticks. Mutations that result in statistically significant decreases in CHIKV infection are colored. Inset, zoomed-in view of the surface representation of Mxra8 at the interface. Side chains of residues of E2 (cyan ribbon) at this interface are displayed as balls and sticks. E. Surface representation of all CHIKV structural protein domains at the interface of Mxra8 at site 3 (shown as ribbon diagram), colored by E2-E1 heterodimer: wrapped, light grey; intraspike, medium grey; interspike, dark grey. CHIKV protein residues are colored magenta if they lose over 30% solvent surface area upon Mxra8 binding as calculated by PDBePISA (http://www.ebi.ac.uk/pdbe/pisa/). F-G. Zoomed-in view of D1 of Mxra8. (F) CHIKV E2 residues identified as epitopes for neutralizing human anti-CHIKV mAbs (Long et al., 2015; Smith et al., 2015) that also block binding to Mxra8 (Zhang et al., 2018) are labeled and colored blue. (G) Four CHIKV E2 residues at the CHIKV-Mxra8 interface as defined by previous alanine scanning mutagenesis data (Zhang et al., 2018) are labeled and colored green. See Fig S2, S4, S5, S7, and Table S4.

Figure 6.. Modes of Mxra8 engagement by CHIKV.

A. Two-dimensional equatorial slice (top panel) and Fourier shell correlation (FSC) plot (bottom panel)of CHIKV infectious particles in complex with Mxra8. B-C. Views of asymmetric units of CHIKV infectious particles (B) or CHIKV VLP (C) with bound Mxra8 electron density at high contour (left panel) and low contour (middle and right panels), colored by structural proteins if within 6 Å of docked model coordinates. Mxra8 coordinates were removed from the model, and E3 was docked in for (B). The value is the standard deviation of density values above the mean. Color scheme: grey, E1; cyan, E2; yellow, E3; capsid, green; Mxra8 and other unexplained density, magenta. D-E. Kinetic sensograms and steady-state analysis of murine Mxra8 binding to CHIKV VLPs fit to a 1:1 binding model (D) or two-site model (one high affinity site and three lower but equal affinity sites) (E). Raw experimental traces are in black, fit traces are in red. Inset, Scatchard plot (4 experiments; mean, standard error of the mean (SEM), and χ² values). See Fig S8 and S9.