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Using Metadynamics to Understand the Mechanism of Calmodulin/Target Recognition at Atomic Detail

G. Fiorin ^*, A. Pastore , P. Carloni ^* and M. Parrinello 

^* International School for Advanced Studies and Democritos Modeling Center for Research in Atomistic Simulation, 34014 Trieste, Italy; Division of Molecular Structure, National Institute for Medical Research, London NW7 1AA, United Kingdom; and Computational Science, Department of Chemistry and Applied Biosciences, ETH Zurich, CH-6900 Lugano, Switzerland

Correspondence: Address reprint requests to P. Carloni, E-mail: carloni{at}sissa.it.

The ability of calcium-bound calmodulin (CaM) to recognize most of its target peptides is caused by its binding to two hydrophobic residues (‘anchors’). In most of the CaM complexes, the anchors pack against the hydrophobic pockets of the CaM domains and are surrounded by fully conserved Met side chains. Here, by using metadynamics simulations, we investigate quantitatively the energetics of the final step of this process using the M13 peptide, which has a high affinity and spans the sequence of the skeletal myosin light chain kinase, an important natural CaM target. We established the accuracy of our calculations by a comparison between calculated and NMR-derived structural and dynamical properties. Our calculations provide novel insights into the mechanism of protein/peptide recognition: we show that the process is associated with a free energy gain similar to that experimentally measured for the CaM complex with the homologous smooth muscle MLCK peptide (Ehrhardt et al., 1995, Biochemistry 34, 2731). We suggest that binding is dominated by the entropic effect, in agreement with previous proposals. Furthermore, we explain the role of conserved methionines by showing that the large flexibility of these side chains is a key feature of the binding mechanism. Finally, we provide a rationale for the experimental observation that in all CaM complexes the C-terminal domain seems to be hierarchically more important in establishing the interaction.

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