Background & Objectives: S100A1 protein is a 10 kDa Ca²⁺ sensor & abundantly expressed in cardiomyocytes (CMs) of vertebrates. Marked reduction in S100A1 level is hallmark of diseased heart, vice versa, addition of S100A1 by gene transfer rescues diseased heart contractile dysfunction. Since our understanding of the molecular circuits that may contribute to the regulation of S100A1 protein levels are still scarce, we conducted a hybrid computational structural & experimental approach to unveil underlying molecular residues & mechanisms that may control S100A1’s protein stability.

Methods & Results: We employed in silico computational prediction & molecular docking tools, respectively, to inform experimental approaches in order to characterize residues within the 94 amino acid (aa) of S100A1. A web server-based GPS-SUMO 2.0 analysis of the human S100A1 sequence unveiled a putative SUMO interacting (SIM) motif (76-VVLVA-80) within the alpha-helical C-terminus of S100A1. Restrained docking with HADDOCK predicted a molecular interaction between SUMO-1 & the SIM-lined groove of the Ca2+-bound (holo) S100A1 homodimer that presents a potential novel type of interaction mode. We then performed an in vitro S100A1-SUMO interaction assay in the presence of 1 mM Ca²⁺ or 1 mM EGTA & the assay revealed calcium dependent specific S100A1-SUMO proteins interaction. Overexpression of S100A1 together with SUMO1 increases S100A1 protein abundance in CMs & COS1 cells without changing the mRNA level of S100A1. Overexpression assays in COS1 cells & CMs involving S100A1 truncation mutant lacking SIM motif (S100A1-1-74) or site directed mutagenesis deleted (DSIM) or Alanine replaced SIM of S100A1 showed that S100A1 lacking SIM motif either via truncation & deletion or Alanine substitution led to, respectively, absence of detection or massively reduced overexpression of S100A1 protein without affecting the mRNA overexpression of the mutants. The aforementioned mutants could be rescued at protein level in CMs & COS1 cells by addition of proteasome inhibitor-MG-132.

Conclusion: Here we describe a yet unrecognized post-translational molecular checkpoint for S100A1’s protein stability involving a SIM-mediated interaction between S100A1 & SUMO.