Determination of the Individual Roles of the Linker Residues in the Inter-Domain Motions of Calmodulin using NMR Chemical Shifts

Abstract

Many protein molecules are formed by two or more domains whose structures and dynamics are closely related to their biological functions. It is challenging, however, to determine the structural properties of these multidomain proteins because of their conformational heterogeneity. Here, we characterize the interdomain motions in the calcium-bound state of calmodulin (Ca2+-CaM) using NMR chemical shifts as replica-averaged structural restraints in molecular dynamics simulations. We find that the conformational fluctuations of the interdomain linker, which are largely responsible for the interdomain motions of CaM, can be well described by exploiting the information provided by chemical shifts, as these parameters are particularly sensitive to the local geometry of polypeptide chains. We thus identify ten residues in the interdomain linker region that change their conformation upon substrate binding. Five of these residues (Met76, Lys 77, Thr79, Asp80 and Ser81) are highly flexible and cover the range of conformations observed in the substrate-bound state, while the remaining five (Arg74, Lys75, Asp78, Glu82 and Glu83) are much more rigid and do not populate conformations typical of the substrate-bound form. The ensemble of conformations representing the Ca2+-CaM state obtained in this study is in agreement with residual dipolar coupling, paramagnetic resonance enhancement and small-angle X-ray scattering measurements, which were not used as restraints in the calculations. These results thus illustrate how CaM binds to its substrates by interdigitated residues in the interdomain linker that follow either the conformational selection or the induced fit mechanisms.