The Effect of Macromolecular Crowding, Ionic Strength and Calcium Binding on Calmodulin Dynamics

The flexibility in the structure of calmodulin (CaM) allows its binding to over 300 target proteins in the cell. To investigate the structure-function relationship of CaM, we combined methods of computer simulation and experiments based on circular dichroism (CD) to investigate the structural characteristics of CaM that influence its target recognition in crowded cell-like conditions. We developed a unique multiscale solution of charges computed from quantum chemistry, together with protein reconstruction, coarse-grained molecular simulations, and statistical physics, to represent the charge distribution in the transition from apoCaM to holoCaM upon calcium binding. Computationally, we found that increased levels of macromolecular crowding, in addition to calcium binding and ionic strength typical of that found inside cells, can impact the conformation, helicity and the EF hand orientation of CaM. Because EF hand orientation impacts the affinity of calcium binding and the specificity of CaM's target selection, our results may provide unique insight into understanding the promiscuous behavior of calmodulin in target selection inside cells. Author Summary: Proteins are workhorses for driving biological functions inside cells. Calmodulin (CaM) is a protein that can carry cellular signals by triggered conformational changes due to calcium binding that alters target binding. Interestingly, CaM is able to bind over 300 targets. One of the challenges in characterizing CaM's ability to bind multiple targets lies in that CaM is a flexible protein and its structure is easily modulated by the physicochemical changes in its surroundings, particularly inside a complex cellular milieu. In order to determine structure-function relationships of CaM, we employed a combined approach of experiments, computer simulations and statistical physics in the investigation of the effect of calcium-binding, salt concentration, and macromolecular crowding on CaM. The results revealed unique folding energy landscapes of CaM in the absence and presence of calcium ions and the structural implications of CaM are interpreted under cell-like conditions. Further, a large conformational change in CaM in response to environmental impacts, dictates the packing of local helices that may be critical to its function of target binding and recognition among vast target selections.