The Role of Protein Dynamics in Calmodulin Target Recognition

Abstract

We aimed to elucidate the role of protein dynamics and stability in target recognition, a process critical in understanding how proteins choose the appropriate target. We chose to examine the ubiquitous protein calmodulin (CaM), an essential secondary messenger of calcium signaling with over 300 identified protein targets. CaM is highly flexible and dynamic and its conformational plasticity is essential for accommodating diverse targets. CaM exists as an ensemble of conformers fluctuating around a large flat energy minimum, where conformational sampling permits a continuum population of structures with similar energies. To investigate the role of conformational sampling we measured CaM properties under conditions of excluded volume, where non-reactive polymers were incorporated into experimental conditions. The polymers effectively limit the 3D space a protein can occupy and thus influence its conformational sampling by redistributing the probabilities of each conformational state and modulating the energy barriers between them. We observed that volume exclusion stabilized compact conformations of CaM which had minimal impact on steady state affinity for Ca2+ or for protein targets. We observed reduced association rates with targets and importantly, volume exclusion significantly decreased the rate of conformational transition from the initial encounter complex to the natively bound complex. This transition is dependent on polymer size, with smaller polymers correlated to larger decreases, as well as temperature, where lower temperatures which impede conformational dynamics decreased rates the most significantly. We conclude that stabilization of compact conformers and dampened protein dynamics induce conformational frustration during transition to the native complex, where sampling of productive transition state intermediates is encumbered by high energy barriers. Our data is in support of an induced fit binding mechanism where CaM and its target must undergo mutually adjusted step-wise conformational searches to find the natively bound complex.