Systematic screening and characterization of novel γ-glutamyl tripeptides binding to the CaSR Venus flytrap domain

The calcium-sensing receptor (CaSR) plays an important role in calcium homeostasis and multiple physiological regulatory processes, and its Venus flytrap (VFT) domain is a key region for γ-glutamyl peptide binding. In this study, molecular docking, surface plasmon resonance (SPR), and molecular dynamics simulations were integrated to systematically investigate the binding behavior of 400 γ-glutamyl tripeptides with CaSR-VFT. The results showed that all 400 γ-glutamyl tripeptides could be accommodated in the central binding pocket of CaSR-VFT, with binding energies mainly ranging from -6 to -10 kcal mol^-1. Interaction site analysis indicated that Ser272, Asn102, Arg66, Ser147, Asn64, and Ser170 were the major high-frequency interacting residues, among which Ser272 showed the highest occurrence frequency. The peptides with the strongest and weakest predicted binding abilities, γEYW and γECF, exhibited docking binding energies of -9.863 and -6.05 kcal mol^-1, respectively. After Ser272Ala mutation, the absolute binding energies of γEYW and γECF decreased by 44.02% and 15.50%, respectively. SPR experiments confirmed that both γEYW and γECF could directly bind to purified CaSR-VFT protein, with KD values of 3.3 μM and 329.4 nM, respectively. Molecular dynamics simulations showed that γECF exhibited a more stable hydrogen-bond network and a more concentrated low-free-energy conformational distribution. Overall, this study screened and validated two novel γ-glutamyl tripeptides capable of binding to CaSR-VFT, providing a reference for the screening of CaSR-targeting peptides and the investigation of their binding mechanisms.