Role of allostery in ligand binding modulated mechanical responses of calmodulin domain

Abstract Calmodulin (CaM), a central Ca2+ sensor in eukaryotic cells, exhibits Ca2+-regulated conformational transitions that are strongly influenced by mechanical stress. Single-molecule experiments have revealed a nonmonotonic dependence of the unfolding transition-state distance δx‡ on Ca2+ concentration, yet the underlying microscopic mechanism remains elusive. Here, using a dynamic energy-landscape model combined with largescale coarse-grained molecular dynamics simulations, we elucidate how Ca2+ concentration and mechanical tension jointly shape the unfolding pathway of CaM domain. At low Ca2+ concentrations, the reduction in δx‡ arises from a redistribution within the folded ensemble between closed and open conformations. At moderate and high Ca2+ concentrations, the subsequent increase in δx‡ originates from substantial Ca2+-dependent modifications of the unfolding transition-state ensemble. Moreover, our simulations predict that mechanical tension enhances Ca2+-binding affinity, revealing a counterintuitive force-induced stabilization effect. Together, these findings provide a microscopic explanation for the experimentally observed tension-Ca2+ coupling and offer a unified physical framework for understanding the mechanosensitive functional regulation of CaM.