Backbone and side chain dynamics of mutant calmodulin-peptide complexes

Allosteric behavior has been found to exist in diverse classes of proteins, such as transport proteins (1), enzymes (2), chemo-mechanical energy transducers (3), receptors (4), chaperonins (5), and signaling proteins (6). Propagation of the allosteric signal is usually associated with the protein undergoing a largely structural response to the binding of ligand to a regulatory site. Nevertheless, it has long been admitted on theoretical grounds that changes in the distribution of thermal fluctuations about the mean structure could accompany a change in functional state of a protein and thereby provide a reservoir of residual entropy that could, in principle, contribute to allosteric phenomena (7–10). Indeed, if a protein is treated as an ensemble of conformational states (11, 12), binding of the allosteric effector could not only stabilize a certain conformation with high affinity for the active site ligand, but could also change the “shape” of the conformational distribution. The latter would be a purely entropic effect that, if significant, would contribute to the free energy of the bound state and manifest itself in a change of the dynamic behavior of the protein. However, although the possibility of allosteric regulation through entropic (dynamic) mechanisms exists, a definitive example of such an allosteric mechanism remains to be fully illuminated (13). The relatively recent experimental demonstration using NMR relaxation methods of the large and heterogeneously distributed residual entropy in proteins (14–16) raises once again the possibility that dynamic effects have been adopted by proteins to carry out allosteric phenomena (13). Indeed, it has been shown in several instances that significant changes in residual protein entropy (dynamics) can result from a change in protein functional state (17, 18). Central to the concept of allostery has been a discussion of pathways of energetic coupling, perhaps most exemplified by the analysis of allosteric regulation in hemoglobin where a highly mechanical model remains a dominant theme (19). The participation of residual protein entropy in allosteric regulation raises the issue of whether pathways of dynamic disorder exist in proteins and whether they have been used to provide free energy transduction. This concept also raises the question of whether dynamically coupled sites need be structurally contiguous in the native structure in order to provide long-range energetic coupling or whether the effects can be generally manifested in the ensemble of states of the protein (12). The basic parameters of coupled dynamics in proteins need to be exposed in order to begin to distinguish these two views of effective long-range energetic coupling. We have initiated such a study with the protein calmodulin. Here we use site-specific mutations as a means for perturbing the conformational ensemble of an allosteric protein and observed the dynamic consequences of these perturbations using solution NMR relaxation techniques. A complex between chicken calmodulin (CaM) and a 21-residue peptide based on the calmodulin-binding domain of the smooth muscle myosin light chain kinase (smMLCKp) was chosen as a model system. CaM is a small acidic protein of 148 amino acids, which binds four Ca2+ ions in a highly cooperative manner ensuring the “all-or-nothing” response to changes in the intracellular calcium levels (20). A unique feature of CaM is that, while being a monomeric protein, it shows two different types of allosteric behavior: (i) cooperativity between calcium binding sites and (ii) cooperativity between its target- and calcium-binding sites. In the absence of calcium ions, smMLCKp binding to CaM is negligible, while in the presence of calcium it forms a very tight complex having a dissociation constant of 1 nM (21). The allosteric coupling between calcium and peptide binding sites is reciprocal, with the Ca2+ binding affinity of CaM increasing 30–100-fold in the presence of smMLCKp (22). Here we examine the extent of variation of the fast dynamics of calmodulin in complex with the smMLCKp domain in response to point mutations that influence calcium- and target domain binding affinity.