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 Å 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 the S100A4−MIIA complex. These studies provide the foundation for understanding S100A4 target recognition and may support the development of reagents that interfere with S100A4 function.