Dynamics of Ca²⁺-saturated calmodulin D129N mutant studied by multiple molecular dynamics simulations

Fifteen independent 1-nsec MD simulations of fully solvated Ca ²⁺ saturated calmodulin (CaM) mutant D129N were performed from different initial conditions to provide a sufficient statistical basis to gauge the significance of observed dynamical properties. In all MD simulations the four Ca²⁺ ions remained in their binding sites, and retained a single water ligand as observed in the crystal structure. The coordination of Ca²⁺ ions in EF-hands I, II, and III was sevenfold. In EF-hand IV, which was perturbed by the mutation of a highly conserved Asp129, an anomalous eightfold Ca²⁺ coordination was observed. The Ca²⁺ binding loop in EF-hand II was observed to dynamically sample conformations related to the Ca²⁺-free form. Repeated MD simulations implicate two well-defined conformations of Ca²⁺ binding loop II, whereas similar effect was not observed for loops I, III, and IV. In 8 out of 15 MD simulations Ca²⁺ binding loop II adopted an alternative conformation in which the Thr62 〉C = O group was displaced from the Ca²⁺ coordination by a water molecule, resulting in the Ca²⁺ ion ligated by two water molecules. The alternative conformation of the Ca²⁺ binding loop II appears related to the "closed" state involved in conformational exchange previously detected by NMR in the N-terminal domain fragment of CaM and the C-terminal domain fragment of the mutant E140Q. MD simulations suggest that conformations involved in microsecond exchange exist partially preformed on the nanosecond time scale.