On the Ca 2+ binding and conformational change in EF-hand domains: Experimental evidence of Ca 2+ -saturated intermediates of N-domain of calmodulin

Double mutation of Q41L and K75I in the N-domain of calmodulin (N-Cam) stabilizes the closed form of N-Cam such that binding of Ca 2+ in solution no longer triggers a conformational change to the open form, and its Ca 2+ binding affinity decreases dramatically. To further investigate the solvation effects on the structure, Ca 2+ binding affinity and conformational dynamics of this N-Cam double mutant in the Ca 2+ saturated state, we solved its X-ray structure. Surprisingly, the structure revealed an open conformation of the domain which contradicts its closed conformation in solution. Here we provide evidence that crystallization conditions were responsible for this Ca 2+ -saturated domain open conformation in the crystal. Importantly, we demonstrate that the presence of the crystallization co-precipitant and alcohols were able to induce a progressive opening of the closed form of this domain, in Ca 2+ saturated state, in solution. However, in the Ca 2+ depleted state, addition of alcohols was unable to induce any opening of this domain in solution. In addition, in the Ca 2+ saturated state, the molecular dynamics simulations show that while N-Cam can populate the open and closed conformation, the N-Cam double mutant exclusively populates the closed conformation. Our results provide experimental evidence of intermediate conformations of Ca 2+ -saturated N-Cam in solution. We propose that conformational change of Ca 2+ sensor EF-hand domains depends on solvation energetics, Ca 2+ binding to promote the full open form, Ca 2+ depleted state conformational dynamics, and the chemical properties of the molecules nearby key residues such as those at positions 41 and 75 in N-Cam.