Experiment and Simulation Reveal Residue Details for How Target Binding Tunes Calmodulin's Calcium-Binding Properties

We aim to elucidate the molecular mechanism of the reciprocal relation of calmodulin's (CaM) target binding and its affinity for calcium ions (Ca²⁺), which is central to decoding CaM-dependent Ca²⁺ signaling in a cell. We employed stopped-flow experiments and coarse-grained molecular simulations that learn the coordination chemistry of Ca²⁺ in CaM from first-principle calculations. The associative memories as part of the coarse-grained force fields built on known protein structures further influence CaM's selection of its polymorphic target peptides in the simulations. We modeled the peptides from the Ca²⁺/CaM-binding domain of Ca²⁺/CaM-dependent kinase II (CaMKII), CaMKIIp (293-310) and selected distinctive mutations at the N-terminus. Our stopped-flow experiments have shown that the CaM's affinity for Ca²⁺ in the bound complex of Ca²⁺/CaM/CaMKIIp decreased significantly when Ca²⁺/CaM bound to the mutant peptide (296-AAA-298) compared to that bound to the wild-type peptide (296-RRK-298). The coarse-grained molecular simulations revealed that the 296-AAA-298 mutant peptide destabilized the structures of Ca²⁺-binding loops at the C-domain of CaM (c-CaM) due to both loss of electrostatic interactions and differences in polymorphic structures. We have leveraged a powerful coarse-grained approach to advance a residue-level understanding of the reciprocal relation in CaM, that could not be possibly achieved by other computational approaches.