Asymmetric mode of Ca²⁺-S100A4 interaction with nonmuscle myosin IIA generates nanomolar affinity required for filament remodeling

Abstract

Filament assembly of nonmuscle myosin IIA (NMIIA) is selectively regulated by the small Ca²⁺-binding protein, S100A4, which causes enhanced cell migration and metastasis in certain cancers. Our NMR structure shows that an S100A4 dimer binds to a single myosin heavy chain in an asymmetrical configuration. NMIIA in the complex forms a continuous helix that stretches across the surface of S100A4 and engages the Ca²⁺-dependent binding sites of each subunit in the dimer. Synergy between these sites leads to a very tight association (K(D) ∼1 nM) that is unique in the S100 family. Single-residue mutations that remove this synergy weaken binding and ameliorate the effects of S100A4 on NMIIA filament assembly and cell spreading in A431 human epithelial carcinoma cells. We propose a model for NMIIA filament disassembly by S100A4 in which initial binding to the unstructured NMIIA tail initiates unzipping of the coiled coil and disruption of filament packing.

Graphical abstract

Figure 1

Interaction of S100A4 with NMIIA (A) Sequence of myosin fragments used in the binding site mapping. M111 corresponds to the whole sequence, M70 is highlighted in blue and red, and M39 in red. Residues in italic identify the unstructured C-terminal region of myosin. Residues in a and d positions of the heptad repeat that are critical for the coiled-coil stability are underlined. (B) Intensities of ¹H,¹⁵N-HSQC cross-peaks of uniformly ¹⁵N-labeled M70 in complex with u-S100A4 normalized on the intensity for the last residue G1938. Low intensities correspond to the residue immobilized by direct contact with S100A4. M39 location is indicated by the red rectangle (C) ¹H,¹⁵N-HSQC spectrum of ¹⁵N-labeled S100A4 in the free form (blue) and in the presence of 0.5 mole equivalent of u-M39 (red). Doubling of the cross-peak demonstrates asymmetric environment for equivalent residues of the S100A4 dimer. Colors of the labels indicate free (blue) and bound (red) forms. Residues are labeled A, B, or A/B to denote the monomer subunit that gives rise to the signal. (D) Gel filtration elution profile of the SEC-MALLS experiment for the 1:1 molar ratio used to determine the molecular weight of S100A4/M111 complex. Both the complex (peak I) and the free form of S100A4 (peak II) are observed. The molecular weight of 39.3 kDa measured for the M111/S100A4 peak is in a very good agreement with 40.1 kDa expected for the 1:2 complex. (E) ITC record of S100A4 titration with M39 at 10 μM S100A4 in the cell and 75 μM of M39 in the syringe. The solid line shows the fitting to the single site-binding model with K_D of 4 nM. See Figure S1 for further binding measurements. See also Table S1.

Figure 2

Structure of M39/S100A4 Complex (A) Superposition of the backbone atoms for ten lowest-energy structures of M39/S100A4 complex. M39 is shown in orange; subunits of S100A4 dimer are in green and cyan. (B) Lowest-energy structure of M39/S100A4 complex in two orientations related by 90° rotation along the vertical axis. The boxed areas indicate sites A and B. (C) Structure of bound M39. Hydrophobic residues of M39 in direct contact with S100A4 are highlighted in magenta and labeled. (D) Hydrophobic contacts at site A (left) and B (right). For clarity only the helices H3 and H4 are shown as viewed from the back in the orientation shown in (B). Helices H3 and H4 are oriented in a similar way for both sites. (E) Superposition of the M39/S100A4 structure on the structures of (from left to right) TRTK-12/S100B (PDB 3IQQ), TRTK-12/S100A1 (PDB 2KBM), and RyRP12/S100A1 (PDB 2K2F). M39 is shown in light orange, S100A4 in green and cyan, S100B and S100A1 in gray, and TRTK-12 and RyRP12 in red and magenta. Note that in contrast to M39/S100A4, each of these complexes is symmetrical and has a 2:2 configuration. Despite this difference, they match some features of the M39/S100A4 complex. TRTK-12 binding to S100B mostly resembles that of the C-terminal nonhelical portion of M39 and the last turn of the helix. TRTK-12 in complex with S100A1 aligns well with the N- and C-terminal ends of the M39 helix. The helix of RyRP12 in the complex with S100A1 is longer and extends further toward the adjacent EF2 site. See Figure S2 for further structural analysis and comparison with other S100 protein complexes.

Figure 3

S100A4 Affects Stress Fibers, Shape, Spreading, and Motility of A431 Cells (A) Cartoon representation of the M39/S100A4 complex illustrating positions of the mutated residues. The two orientations differ by a 90° rotation along the horizontal axis. Zoomed region shows contacts of the mutated residues V77 and C81 (side chains are shown as sticks; residues are highlighted in yellow). M39 is shown in orange; side chains contacting V77 and C81 in the complex are presented as sticks and labeled. (B) V77D and C81D mutants have impaired interaction with NMIIA. S100A4-containing complexes were immunoprecipitated from lysates of nucleofected cells. Myosin IIA and S100A4 were detected by western blotting. As a control for loading, 5% of the input lysates were run on the gel. See Figure S3 for further characterization of binding of mutant S100A4. (C) WT but not mutant S100A4 activates cell migration in transwell assay. Migration rates were expressed as the ratio of the averaged number of migrating cells relative to the control that was set at 1. Values represent averages of three independent experiments each performed in triplicate. Error bars are SDs among three independent experiments. (D) Cell spreading on the collagen-coated micropatterned glass. Nucleofected cells, pBI empty vector (Control), or pBI vector expressing WT or S100A4 mutant (C81D) were seeded on the collagen type I micropatterned equilateral triangles (length, 41 μm). Cells were stained with antibodies against myosin IIA or S100A4. (E–G) Analyses were performed using images from 50 cells stained for myosin IIA and S100A4 as in (C). Error bars represent SDs between measurements of 50 cells. ^∗p < 0.05 versus control; ^∗∗p < 0.005 and ^∗∗∗p < 0.0001. (E) Cell shape analysis. An area of a fully spread cell that adopted the maximum size of the collagen-coated equilateral triangle micropattern was set at 100% (see Figure S3). (F) Relative area analysis. The area of a spread cell was calculated as the percent relative to the triangle pattern area, 841 μm. (G) Comparative analysis of the peripheral stress fiber formation. Relative length of the stress fibers along the cells was measured and plotted against cell perimeter, where the perimeter was set at 100%.

Figure 4

Effect of S100A4 on the Coiled-Coil Structure of Myosin (A and B) Negatively stained fields of S100A4 alone and M200/S100A4 complex. Black arrowheads and arrows point to individual molecules in each field. (C and D) Representative averaged images of S100A4 alone and M200/S100A4 complex, respectively. Each averaged image contains 30–50 images. (E) Fitting of S100A4 and coiled-coil tail atomic models to M200/S100A4 averaged image. Left panel is a selected averaged image of M200/S100A4 (taken from asterisk-marked average in D). Middle panel is an equivalent view of assembled atomic models to the average. Right panel is a superposition of equivalent view of the atomic model on the average. The lowest-energy structure of the complex was used to model M39/S100A4; coiled-coil myosin model was based on the structure of myosin V (PDB 2DFS). White arrowheads in the left panel indicate the region of S100A4 in M200/S100A4 and the emergence of the M200 densities from the S100A4. Scale bars apply to the field in (A) and (B) (50 nm) and averaged images of class images in (C) and (D) (25 nm).

Figure 5

S100A4 Interaction with Myosin Filaments (A) Schematic diagram of the assembled filament illustrating staggered packing of myosin units. (B) Model of the C-terminal region of the myosin coiled coil (top) and myosin-S100A4 complex (bottom). Side-chain atoms of the residues in positions a and d of the heptad repeat that are crucial for the coiled-coil stability shown as spheres. Large hydrophobic residues that stabilize coiled coil are highlighted in blue; nonoptimal destabilizing residues are in red. S100A4-binding site in myosin is colored in orange and the rest of the myosin in gray. S100A4 subunits are shown in pale green and pale blue. A patch of nonoptimal residues precedes the S100A4-binding site. (C) Schematic diagram of the S100A4 interaction with myosin showing possible intermediates. Subunits of the S100A4 dimer are shown in red and blue. Initially, S100A4 may interact with the unstructured region that is likely to be accessible in the filaments. These sites could engage separate S100A4 dimers or simultaneously bind to the two EF2 sites of a single dimer. The binding of the two dimers to the unstructured region of the coiled coil could be further stabilized by the tetramer formation detected in the crystal structure of S100A4 (Gingras et al., 2008). Potentially, all three intermediate states could exist in equilibrium. (D) Sequence comparison of the S100A4-binding site in NMAIIA with the corresponding region in NMAIIB. The residues involved in the direct contact with S100A4 are marked with an asterisk (“^∗”). Identical residues are highlighted in orange, similar in green. Chemically distinct residues are shown in black.