Calmodulin Conformational Binding Entropy Is Driven by Transient Salt Bridges

Abstract

Calmodulin (CaM) is a calcium-binding protein that mediates signal transduction through an ability to differentially bind to highly variable binding sequences in target proteins. Experimental measurements show a linear correlation between conformational entropy and overall binding entropy, suggesting that a predictive ability to calculate conformational entropy is critical to the identification of binding partners and their relative affinities. To identify how binding affects low- and high-frequency motions, and their relationships to conformational entropy, we have employed fully atomistic molecular dynamics simulations with explicit solvent. The calculated quasiharmonic entropies of CaM bound to the targets relative to unbound CaM are in agreement with experimentally derived conformational entropies, where extrapolation to infinite simulation time showed that at least 92% of the backbone entropy is captured over the course of the 100 ns simulation. Enhanced side chain entropy results from conformational disruption of the protein backbone within loop regions between sub-domain elements, acting to facilitate the formation of transient salt bridges between Lys148 at the C-terminus and GLU sidechains both in CaM helix A and GLU side-chains present in selected binding sequences (i.e., endothelial and neuronal nitric oxide synthases). Comparison of Cα root-mean-squared fluctuations and MET sidechain dihedral distributions of CaM bound to each of the targets revealed that MET sidechains have more torsional freedom when the backbone is more flexible, showing that MET sidechain rotation mirrors the overall conformational binding entropy. Taken together, these results help to illuminate the interplay between electrostatic, hydrophobic, backbone and sidechain properties in the ability of CaM to recognize and discriminate against targets by tuning its conformational entropy.