BACKGROUND: Tryptophan-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. METHODS: The 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. RESULTS: We 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. CONCLUSIONS: This 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 SIGNIFICANCE: The 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.
BACKGROUND:Tryptophan-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. METHODS: The 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. RESULTS: We 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. CONCLUSIONS: This is the first study demonstrating that the vasoactive dipeptideTrp-His possesses inhibitory activity against Ca²⁺-CaM complex formation, which may elucidate how Trp-His inhibited CaMKII in a previous study. GENERAL SIGNIFICANCE: The 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.
Authors: Innocent U Okagu; Timothy P C Ezeorba; Rita N Aguchem; Ikenna C Ohanenye; Emmanuel C Aham; Sunday N Okafor; Carlotta Bollati; Carmen Lammi Journal: Int J Mol Sci Date: 2022-07-30 Impact factor: 6.208