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 Ca 2+ -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 Ca 2+ 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 Ca 2+ modifies the overall organization and structure of the protein, we determined the 1.7 Å crystal structure of the human Ca 2+ -S100A4. Ca 2+ 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 Ca 2+ -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 Ca 2+ 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 Ca 2+ -S100A4. Most of these residues are not exposed in apo-S100A4 and explain the Ca 2+ 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. S100A4, also called mts1, is a member of the S100 family of small, homodimeric, EF-hand Ca 2+ binding proteins. S100 proteins are expressed in a tissue specific manner and bind to a variety of target proteins, resulting in the regulation of specific cellular processes, including cell-cycle regulation, protein phosphorylation, cell growth, motility, differentiation, and survival (1-4). While S100A4 is expressed in a wide range of normal tissues (5,6), it is recognized that an increased level of S100A4 expression correlates with a high incidence of metastasis and poor prognosis for cancer patients (7,8). High S100A4 expression levels are associated with several metastatic cancers, including breast (9), colorectal (10), bladder (11), † This work was supported by National Institutes of Health Grants GM069945 (A.R.B.), GM58888 (D.J.W.), and CA107331 (D.J. W.) and American Cancer Society Grant CDD107745 (D.J.W.). * To whom correspondence should be addressed: A.R.B.: telephone, (718) 430-2741; fax, (718) 430-8565; e-mail, bresnick@aecom.yu.edu. D.J.W.: telephone, (410) 706-4354; fax, (410) 706-0458; e-mail, dweber@umaryland.edu. S.C.A.: telephone, (718) 430-2746; fax, (718) 430-8565; e-mail, almo@aecom.yu.edu. § These authors contributed equally to the completion of this work. ⊥ Current address: Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, U.K. NIH Public Access Author ManuscriptBiochemistry. Auth...

Crystal structure of the S100A4–nonmuscle myosin IIA tail fragment complex reveals an asymmetric target binding mechanism

“…Because previous studies on the NMIIA-S100A4 interaction have not unambiguously determined the myosin sequence requirements for S100A4 binding (16,35,36), isothermal titration calorimetric (ITC) measurements were carried out with peptides and fragments illustrated in S100A4 than it was previously reported (16) (Fig. S1A and Table S1).…”

“…An example of such a structural element is loop 2, which is able to bind both the hydrophilic N terminus and the hydrophobic C terminus of MPT. Conformational adaptation is also extensive when we compare the peptide-free, Ca 2þ -bound wild-type S100A4 (16,17) with the mutant S100A4 protein in complex with MPT (Fig. S6).…”

“…Although the structural transition on binding Ca 2þ is well characterized, atomic details of S100A4 binding to any of its partners have not been described yet. In two crystal structure of Ca 2þ -bound S100A4 the hydrophobic cleft of each subunit is occupied by the C terminus of an adjacent dimer (16,17), which could explain the formation of S100A4 oligomers that were observed extracellularly (18). These clefts were also shown to interact with the calmodulin antagonist trifluoperazine (19).…”

“…S100A4 binds to the C-terminal end of the coiled-coil tail of NMIIA (29) overlapping the assembly competence domain (ACD) that is required for filament formation (30)(31)(32)(33)(34). Despite several efforts to map the S100A4 binding site on NMIIA (16,35,36), the observations are ambiguous, and until now there is no detailed structural model for how the S100A4 dimer dissociates NMIIA filaments. Here, we report the high-resolution crystal structure of the S100A4-NMIIA fragment complex, which reveals a unique target-recognizing mechanism in the S100 family.…”

Phenothiazines inhibit S100A4 function by inducing protein oligomerization

“…The basic organization of the S100 proteins is a symmetric, antiparallel homodimer, in which the N-and C-terminal helices (helices 1 and 4) from each subunit interact to form a stable four helix bundle that serves as the dimer interface. Calcium binding to the C-terminal typical EF-hand significantly alters the angle between helices 3 and 4, which flank the C-terminal Ca 2þ -binding loop, and exposes a hydrophobic cleft that constitutes a binding surface for target proteins (3)(4)(5). Thus the S100 proteins operate as calcium-activated switches that bind and regulate the activity of diverse protein targets.…”