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S100A1, a 21-kDa dimeric Ca 2؉-binding protein, is an enhancer of cardiac Ca 2؉ release and contractility and a potential therapeutic agent for the treatment of cardiomyopathy. The role of S100A1 in skeletal muscle has been less well defined. Additionally, the precise molecular mechanism underlying S100A1 modulation of sarcoplasmic reticulum Ca 2؉ release in striated muscle has not been fully elucidated. Here, utilizing a genetic approach to knock out S100A1, we demonstrate a direct physiological role of S100A1 in excitation-contraction coupling in skeletal muscle. We show that the absence of S100A1 leads to decreased global myoplasmic Ca 2؉ transients following electrical excitation. Using high speed confocal microscopy, we demonstrate with high temporal resolution depressed activation of sarcoplasmic reticulum Ca 2؉ release in S100A1 ؊/؊ muscle fibers. Through competition assays with sarcoplasmic reticulum vesicles and through tryptophan fluorescence experiments, we also identify a novel S100A1-binding site on the cytoplasmic face of the intact ryanodine receptor that is conserved throughout striated muscle and corresponds to a previously identified calmodulin-binding site. Using a 12-mer peptide of this putative binding domain, we demonstrate low micromolar binding affinity to S100A1. NMR spectroscopy reveals this peptide binds within the Ca 2؉ -dependent hydrophobic pocket of S100A1. Taken together, these data suggest that S100A1 plays a significant role in skeletal muscle excitation-contraction coupling, primarily through specific interactions with a conserved binding domain of the ryanodine receptor. This warrants further investigation into the use of S100A1 as a therapeutic target for the treatment of both cardiac and skeletal myopathies.The S100 family of proteins, so named because of their solubility in 100% ammonium sulfate, are small (16 -26 kDa), acidic, Ca 2ϩ

In heart and skeletal muscle an S100 protein family member, S100A1, binds to the ryanodine receptor (RyR) and promotes Ca 2؉ release. Using competition binding assays, we further characterized this system in skeletal muscle and showed that Ca 2؉ -S100A1 competes with Ca 2؉ -calmodulin (CaM) for the same binding site on RyR1. In addition, the NMR structure was determined for Ca 2؉ -S100A1 bound to a peptide derived from this CaM/S100A1 binding domain, a region conserved in RyR1 and RyR2 and termed RyRP12 (residues 3616 -3627 in human RyR1). Examination of the S100A1-RyRP12 complex revealed residues of the helical RyRP12 peptide (Lys-3616, Trp-3620, Lys-3622, Leu-3623, Leu-3624, and Lys-3626) that are involved in favorable hydrophobic and electrostatic interactions with Ca 2؉ -S100A1. These same residues were shown previously to be important for RyR1 binding to Ca 2؉ -CaM. A model for regulating muscle contraction is presented in which Ca 2؉ -S100A1 and Ca 2؉ -CaM compete directly for the same binding site on the ryanodine receptor.Excitation coupling is a process by which sarcolemmal depolarization triggers Ca 2ϩ release from the sarcoplasmic reticulum (SR), 4 leading to Ca 2ϩ activation of the thin filaments and muscle fiber contraction. The ryanodine receptor (RyR1) Recently, several studies demonstrated that an S100 protein, S100A1, enhances RyR1-and RyR2-dependent calcium release in both skeletal and cardiac muscle, respectively (5-10). Specifically, S100A1 knock-out skeletal muscle fibers demonstrate decreased Ca 2ϩ transients (6), and adenoviral delivery of S100A1 into failing cardiomyocytes restores myocyte contractile properties (11). Additionally, S100A1 increases [ 3 H]ryanodine binding to RyR1, indicative of increased activation of the channel (5), and S100A1 binds directly to RyR1 in a calciumdependent manner (6). These data suggest a possible therapeutic role of S100A1 in treatment strategies for skeletal and cardiomyopathies (6,8,11). S100A1 is a symmetric homodimer (93 residues/subunit) with each S100A1 subunit having a low affinity pseudo-EF hand and a second high affinity canonical EF hand calcium binding domain (12). The solution structures of apo-and Ca 2ϩ -S100A1 were solved previously using NMR methods (12, 13), and show that a large reorientation of helix 3 occurs in S100A1 upon the addition of calcium. This conformational change exposes a hydrophobic pocket on each S100A1 subunit (12,14), providing a binding site for target proteins such as RyR1 and RyR2. Here we show that a 12-residue peptide (termed RyRP12), derived from the CaM/S100A1-binding site on both RyR1 and RyR2, interacts with a major portion of the target protein-binding site on Ca 2ϩ -S100A1 (6, 15, 16). We present the solution NMR structure of RyRP12 bound to Ca 2ϩ -S100A1, which has several striking similarities to that observed previously for the RyR1 (residues 3614 -3643 in human)-CaM complex (17). Furthermore, competition binding experiments show that Ca 2ϩ -S100A1 competes directly with an RyR antagonist, Ca 2ϩ -CaM, for...

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...