Structure of Ca2+-bound S100A4 and its interaction with peptides derived from nonmuscle myosin-IIA

Abstract

S100A4, also known as mts1, is a member of the S100 family of Ca2+-binding proteins that is directly involved in tumor invasion and metastasis via interactions with specific protein targets, including nonmuscle myosin-IIA (MIIA). Human S100A4 binds two Ca2+ ions with the typical EF-hand exhibiting an affinity that is nearly 1 order of magnitude tighter than that of the pseudo-EF-hand. To examine how Ca2+ modifies the overall organization and structure of the protein, we determined the 1.7 A crystal structure of the human Ca2+-S100A4. Ca2+ binding induces a large reorientation of helix 3 in the typical EF-hand. This reorganization exposes a hydrophobic cleft that is comprised of residues from the hinge region,helix 3, and helix 4, which afford specific target recognition and binding. The Ca2+-dependent conformational change is required for S100A4 to bind peptide sequences derived from the C-terminal portion of the MIIA rod with submicromolar affinity. In addition, the level of binding of Ca2+ to both EF-hands increases by 1 order of magnitude in the presence of MIIA. NMR spectroscopy studies demonstrate that following titration with a MIIA peptide, the largest chemical shift perturbations and exchange broadening effects occur for residues in the hydrophobic pocket of Ca2+-S100A4. Most of these residues are not exposed in apo-S100A4 and explain the Ca2+ dependence of formation of theS100A4-MIIA complex. These studies provide the foundation for understanding S100A4 target recognition and may support the development of reagents that interfere with S100A4 function.

Figure 1

Thermodynamic data showing Ca²⁺ binding to S100A4. (A) Isothermal titration calorimetry (ITC) data demonstrating the interaction of S100A4 with Ca²⁺ (n = 2). (B) Competition assay with the chelator 5,5′Br₂-BAPTA to examine the affinity of S100A4 for Ca²⁺ in the presence of MIIA^1851–1960. The decrease in absorbance was monitored at 263 nm for a mixture containing 25 μM 5,5′Br₂-BAPTA, 12.5 μM S100A4 dimer, and 125 μM MIIA^1851–1960. The inset shows the saturation curve representation for the best fit in Caligator.

Figure 2

Chemical shift perturbations following the addition of Ca²⁺ to apo-S100A4. (A) Bar graph of the cumulative ¹H, ¹⁵N, and ¹³C chemical shift perturbations observed per residue upon the addition of Ca²⁺ to apo-S100A4. (B) Ribbon diagram of Ca²⁺-S100A4 showing the subunits in red and blue. Residues highlighted in yellow exhibited the largest perturbations (as per the dashed red line at 900 Hz in panel A).

Figure 3

(A) Heavy atom positions detected by SHELXD based on the SAD signal at 1.7 Å wavelength. Green and orange spheres denote Ca²⁺ ions and sulfur atoms, respectively. (B) Refined model of S100A4 (residues Ala2-Gly21) superimposed onto the 2F_o – F_c electron density map, contoured at 1σ.

Figure 4

Molecular architecture of the Ca²⁺-S100A4 dimer. (A) Ribbon diagram of the dimer, where the two subunits are colored blue and red. The key structural elements common for the S100 family are indicated. Green spheres denote Ca²⁺ ions. (B) Details of the dimer interface of S100A4 involving helix 1–1′, 4–4′, and 1–4′ (1′–4) contact areas.

Figure 5

Structures of the Ca²⁺-loaded S100A4 EF-hands. (A) N-Terminal pseudo-EF-hand. The Ca²⁺ ion is coordinated by the main chain carbonyl oxygens of Ser20, Glu23, Asp25, and Lys28; the side chain carboxylate of Glu33 (both oxygens), and a water molecule. (B) C-Terminal typical EF-hand. The Ca²⁺ ion is coordinated by the side chains of Asp63, Asn65, Asp67, and Glu74 (both oxygens); the carbonyl oxygen of Glu69; and a water molecule. Green and red spheres denote Ca²⁺ and water atoms, respectively.

Figure 6

Conformational rearrangements in S100A4 caused by Ca²⁺ binding. (A) Ribbon diagrams comparing the three-dimensional structures of apo-S100A4 (red) and Ca²⁺-S100A4 (blue). Green spheres denote Ca²⁺ ions. Stereoviews of the (B) pseudo-EF-hand and (C) typical EF-hand of apo-S100A4 (beige) and Ca²⁺-S100A4 (blue). The Ca²⁺-coordinating oxygen atoms are colored red in both the apo and Ca²⁺-bound states.

Figure 7

Self-association of S100A4 molecules in the crystal lattice. (A) The S100A4 dimer is shown with one red and one blue subunit. The symmetry-related molecules (gray) in the S100A4 crystals are positioned such that their C-terminal tails (green) bind to the hydrophobic cleft of the central molecule. (B) The interacting S100A4 dimers form infinite superhelical structures in the crystal.

Figure 8

Interaction of the C-terminal tail of S100A4 (green) with the ligand binding site of the symmetry-related S100A4 molecule (gray). (A) Overall view of the ligand binding pocket. Residues showing chemical shift perturbations upon binding a peptide derived from the C-terminus of S100A4 (residues Glu88–Lys101) are colored orange. (B) Zoomed in view. Many of the interactions are nonpolar; however, Glu91 and Asp95 of the peptide may form ionic interactions with Arg49, Asn61, and Lys57 from the symmetry-related molecule.

Figure 9

Chemical shift perturbations of ¹H–¹⁵N correlations in a 2D HSQC spectrum of Ca²⁺-S100A4 upon the addition of a peptide derived from the C-terminus of S100A4. (A) HSQC spectra of Ca²⁺-S100A4 in the absence (black contours) and presence of 0.6 mM (red contours) and 3.6 mM (blue contours) C-terminal S100A4 peptide. The inset shows an expanded region of the HSQC spectrum illustrating the perturbations to the ¹H–¹⁵N correlation for F55. (B) Chemical shift perturbations for residues in Ca²⁺-S100A4 upon addition of 3.6 mM C-terminal S100A4 peptide.

Figure 10

Sedimentation equilibrium measurements taken at 25 °C. The solid lines represent the best fit from a global nonlinear least-squares analysis of data obtained at 15000 and 22000 rpm: (□) 38 μM wild-type S100A4 and CaCl₂ (subunit concentration), (○) 54 μM wild-type S100A4 and EDTA/EGTA (subunit concentration), and (◇) 61 μM Δ13C S100A4 and CaCl₂ (subunit concentration).

Figure 11

Thermodynamic data showing MIIA peptide binding to S100A4. (A) Isothermal titration calorimetry (ITC) of MIIA^1893–1923 peptide (low salt; n = 1). (B) Competition fluorescence anisotropy of MIIA^1893–1923 and MIIA^1851–1960 at a physiological salt concentration.

Figure 12

Nuclear magnetic resonance (NMR) data together with the resonance assignments for Ca²⁺-S100A4 in the presence and absence of the MIIA^1908–1923 peptide. (A) Strips from ¹⁵N planes of a three-dimensional HNCACB data set for ¹³C- and ¹⁵N-labeled S100A4 bound to the MIIA^1908–1923 peptide in the presence of Ca²⁺, which illustrates the quality of the data used for making the resonance assignments. (B) Overlay of ¹H–¹⁵N HSQC data for Ca²⁺-S100A4 in the MIIA^1908–1923 peptide-bound (red contours) and free (black contours) states.

Figure 13

Ribbon and surface diagrams of S100A4 in the (A) apo and (B) Ca²⁺-bound states. Residues colored yellow show significant chemical shift perturbations and exchange broadening effects upon binding of the MIIA^1908–1923 peptide.