Inhibition of calcium-calmodulin complex formation by vasorelaxant basic dipeptides demonstrated by in vitro and in silico analyses

•Trp-His is a new CaM inhibitor inhibiting CaM from forming a complex with Ca2 +.•A peptide skeleton and a basic amino acid are the crucial structural features.•His, N hydrogen atom in imidazole ring, and Trp are also important for CaM binding.•Trp-His binds to Ca2 +-binding site on CaM with a stoichiometry of 1:1.•Trp-His (protonated form) forms hydrogen bonds with CaM with ∆Gbind of − 49.1 kJ/mol.

BackgroundTryptophan-histidine (Trp-His) was found to suppress the activity of the Ca2 +/calmodulin (CaM)-dependent protein kinases II (CaMKII), which requires the Ca2 +-CaM complex for an initial activation. In this study, we attempted to clarify whether Trp-His inhibits Ca2 +-CaM complex formation, a CaMKII activator.MethodsThe ability of Trp-His and other peptides to inhibit Ca2 +-CaM complex formation was investigated by a Ca2 +-encapsulation fluorescence assay. The peptide-CaM interactions were illustrated by molecular dynamic simulation.ResultsWe showed that Trp-His inhibited Ca2 +-CaM complex formation with a 1:1 binding stoichiometry of the peptide to CaM, considering that Trp-His reduced Hill coefficient of Ca2 +-CaM binding from 2.81 to 1.92. His-Trp also showed inhibitory activity, whereas Trp + His, 3-methyl His-Trp, and Phe-His did not show significant inhibitory activity, suggesting that the inhibitory activity was due to a peptide skeleton (irrespective of the sequence), a basic amino acid, a His residue, the N hydrogen atom of its imidazole ring, and Trp residue. In silico studies suggested the possibility that Trp-His and His-Trp interacted with the Ca2 +-binding site of CaM by forming hydrogen bonds with key Ca2 +-binding residues of CaM, with a binding free energy of − 49.1 and − 68.0 kJ/mol, respectively.ConclusionsThis is the first study demonstrating that the vasoactive dipeptide Trp-His possesses inhibitory activity against Ca2 +-CaM complex formation, which may elucidate how Trp-His inhibited CaMKII in a previous study.General significanceThe results provide a basic idea that could lead to the development of small peptides binding with high affinity to CaM and inhibiting Ca2 +-CaM complex formation in the future.

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