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. 2014 Jan 7;22(1):136-48.
doi: 10.1016/j.str.2013.10.013. Epub 2013 Dec 5.

Structure and self-assembly of the calcium binding matrix protein of human metapneumovirus

Cedric Leyrat  1 , Max Renner  1 , Karl Harlos  1 , Juha T Huiskonen  1 , Jonathan M Grimes  2
Affiliations

Structure and self-assembly of the calcium binding matrix protein of human metapneumovirus

Cedric Leyrat et al. Structure. .

Abstract

The matrix protein (M) of paramyxoviruses plays a key role in determining virion morphology by directing viral assembly and budding. Here, we report the crystal structure of the human metapneumovirus M at 2.8 Å resolution in its native dimeric state. The structure reveals the presence of a high-affinity Ca²⁺ binding site. Molecular dynamics simulations (MDS) predict a secondary lower-affinity site that correlates well with data from fluorescence-based thermal shift assays. By combining small-angle X-ray scattering with MDS and ensemble analysis, we captured the structure and dynamics of M in solution. Our analysis reveals a large positively charged patch on the protein surface that is involved in membrane interaction. Structural analysis of DOPC-induced polymerization of M into helical filaments using electron microscopy leads to a model of M self-assembly. The conservation of the Ca²⁺ binding sites suggests a role for calcium in the replication and morphogenesis of pneumoviruses.

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Figures

Figure 1
Figure 1
Crystal Structure of HMPV M (A) Structure of the M dimer. One of the monomers is colored from blue (N terminus) to red (C terminus) with secondary structure elements labeled, whereas the other one is in gray. (B) Close-up of the Ca2+ binding site. The Ca2+ ion is represented as a purple sphere in the electron density from an omit map (contour level = 4σ) calculated using PHASER and omitting the Ca2+ ion. Coordinating water molecules are displayed as nonbonded spheres. See also Figure S1.
Figure 2
Figure 2
Small-Angle X-Ray Scattering (A) Fitted SAXS profiles of the SUMO-3C-M/untagged M mixture (black spheres) and merged data (gray spheres), measured in 1M non-detergent sulfobetaines 201 (NDSB-201) buffer, and SAXS profile of untagged M in 1M NaCl buffer (green spheres). The red lines represent fits from the OEs. (B) Radius of gyration (Rg) distributions of the initial pool of 11,000 models (black area), composed of models of untagged M and models bearing one or two N-terminal SUMO-3C tags, and OEs for the SUMO-3C-M/untagged M mixture (gray line) and untagged M (green line). (C and D) Views of five superimposed representative models from the OE corresponding to the SUMO-3C-M/untagged M mixture measured in 1M NDSB-201 buffer. Residues missing from the crystal structure are shown in red, or in orange for residues that are missing in only one monomer. (E and F) Similar views of the OE from untagged M in 1M NaCl. See also Figures S2 and S3.
Figure 3
Figure 3
Fluorescence-Based TSA (A) Close-up of the second (low-affinity) Ca2+ binding site in the crystal structure. (B) Model of binding of Ca2+ as observed in MDS. (C) Unfolding transitions of native SUMO-3C-M (black) in 50 mM HEPES, pH 7.8, and 1.15 M NaCl and with increasing concentrations of EGTA (indicated by an arrow). Only the titration performed using 4 μM of protein is represented for clarity. (D) Plots of Tm versus total EGTA concentration, for a protein concentration of 4 μM (black) or 2 μM (red). The data points were fitted using a four-parameter sigmoidal dose-response function. (E) Unfolding transitions of native SUMO-3C-M (black) in 50 mM HEPES, pH 7.8, and 1.15 M NaCl and with increasing concentrations of CaCl2 from 0.25 mM to 2 mM (indicated by an arrow). (F) Plot of ΔΔGu versus total Ca2+ concentration. ΔΔGu values were calculated and fitted using equations from Layton and Hellinga (2010). See also Figure S4.
Figure 4
Figure 4
Helical Ordering of M in the Presence of Lipids Visualized by Electron Microscopy (A) Samples of M incubated in the presence of DOPC stained with uranyl acetate revealed tubular and spherical structures with free M in the background. Scale bar = 100 nm. (B) A close-up of free M dimers. Inset shows a class average of M calculated from 577 of 840 particles. (C) A close-up of a tubular filament. M is seen coating the filament surface. (D) A close-up of an M-coated spherical structure. (E) A computationally straightened, long tubule. Scale bars in (B)–(E) = 25 nm. (F) A computational diffraction pattern of the tubule shown in (E) reveals maxima on layer lines, indicating that the tubule has helical symmetry. Lattice indexes (white numbers) and layer line heights (black numbers) are indicated for clearly visible maxima. (G) A radially depth-cued isosurface representation of the density map for lipid-bound M is shown. The map was calculated using helical reconstruction from electron microscopy images of negatively stained samples. (H) A close-up of the map (gray transparent surface) shows the packing of the fitted M (blue) after imposing helical symmetry. Only the C-alpha trace is shown for M. (I) Same rendering as (H), but shown from the side. All isosurfaces were calculated at 2σ above the mean value. See also Figure S5.
Figure 5
Figure 5
Structural Alignments and Evolutionary Relationships between M Proteins from Paramyxoviridae, Bornaviridae, and Filoviridae (A) Structure-based unrooted phylogenetic tree of known M structures from HMPV, RSV, NDV, EBOV, and BDV. (B) Structure-based unrooted phylogenetic tree of M protein NTD and CTD domains. Evolutionary information was calculated by structural alignment using SHP (Abrescia et al., 2012) and plotting was done using PHYLIP. See also Figure S6.
Figure 6
Figure 6
Comparison of the Quaternary Structures of M from HMPV, NDV, and BDV The structures are shown in cartoon and colored by chain. Electrostatic surfaces were drawn using vacuum charges in Pymol.
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