Functional Calmodulin States Are Selected from an Electrostatically Tuned Free Energy Landscape

Calmodulin (CaM) is a versatile calcium-binding protein whose structural flexibility enables regulation of diverse cellular processes, but capturing its full conformational landscape remains challenging due to high energy barriers between states. Here we employ well-tempered metadynamics simulations using physically interpretable collective variables to explore CaM conformations under calcium-bound and calcium-free states at physiological and low salt concentrations. We identify four principal conformations that shift in population depending on calcium binding and ionic strength. Calcium binding favors compact states, while low salt conditions flatten the energy landscape, facilitating transitions, but also causing kinetic trapping due to salt-bridge interactions. Comparison with experimental CaM-protein complexes shows that target binding stabilizes extended conformations distinct from the minima accessible to free CaM, illuminating how calcium and the ionic environment orchestrate CaM's conformational dynamics in cellular signaling.