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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...

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SummaryAs is typical for S100-target protein interactions, a Ca 2+ -dependant conformational change in S100A1 is required to bind to a 12-residue peptide (TRTK12) derived from the actin capping protein, CapZ. In addition, the Ca 2+ -binding affinity of S100A1 is found to be tightened (> 3-fold) when TRTK12 is bound. To examine the biophysical basis for these observations, the solution NMR structure of TRTK12 in a complex with Ca 2+ -loaded S100A1 was determined. When bound to S100A1, TRTK12 forms an amphipathic helix (residues N6 to S12) with several favorable hydrophobic interactions observed between W7, I10, and L11 of the peptide and a well-defined hydrophobic binding pocket in S100A1 that is only present in the Ca 2+ -bound state. Next, the structure of S100A1-TRTK12 was compared to that of another S100A1-target complex (i.e. S100A1-RyRP12), which illustrated how the binding pocket in Ca 2+ -S100A1 can accommodate peptide targets with varying amino acid sequences. Similarities and differences were observed when comparing the structures of S100A1-TRTK12 and S100B-TRTK12, providing insights regarding how more than one S100 protein can interact with the same peptide target. Such comparisons, including those to other S100-target and S100-drug complexes, provide the basis for designing novel small molecule inhibitors that could be specific for blocking one or more S100-target protein interaction(s).