Abstract
Protein-protein interactions are often mediated by flexible loops that experience conformational dynamics on the microsecond to millisecond time scales. NMR relaxation studies can map these dynamics. However, defining the network of inter-converting conformers that underlie the relaxation data remains generally challenging. Here, we combine NMR relaxation experiments with simulation to visualize networks of inter-converting conformers. We demonstrate our approach with the apo Pin1-WW domain, for which NMR has revealed conformational dynamics of a flexible loop in the millisecond range. We sample and cluster the free energy landscape using Markov State Models (MSM) with major and minor exchange states with high correlation with the NMR relaxation data and low NOE violations. These MSM are hierarchical ensembles of slowly interconverting, metastable macrostates and rapidly interconverting microstates. We found a low population state that consists primarily of holo-like conformations and is a “hub” visited by most pathways between macrostates. These results suggest that conformational equilibria between holo-like and alternative conformers pre-exist in the intrinsic dynamics of apo Pin1-WW. Analysis using MutInf, a mutual information method for quantifying correlated motions, reveals that WW dynamics not only play a role in substrate recognition, but also may help couple the substrate binding site on the WW domain to the one on the catalytic domain. Our work represents an important step towards building networks of inter-converting conformational states and is generally applicable.
Highlights
Protein-protein interactions are often mediated by flexible motifs or domains that make conformational transitions on slow time scales
We present a novel way to use Nuclear Magnetic Resonance, Molecular Dynamics Simulations, and Markov State Models to uncover a map of this plethora of conformations that is consistent with the available data
We applied this method to study the intrinsic dynamics used in substrate binding by the WW domain of the Pin1 proline cis-trans isomerase and found that the Nuclear Magnetic Resonance (NMR) data were best explained by two slowly-interconverting sets of many metastable conformations rather than two distinct macrostates
Summary
Protein-protein interactions are often mediated by flexible motifs or domains that make conformational transitions on slow (ms{ms) time scales. Nuclear Magnetic Resonance (NMR) relaxation experiments have emerged as a premier tool for revealing the location and time scale of these transitions [2,3]. It is reasonable to turn to molecular dynamics simulations. Such simulations can retain all molecular degrees of freedom, and offer conformational ensembles that may be evaluated on the basis of their consistency with experiment. Current NMR spin relaxation experiments can reveal microsecond-millisecond conformational dynamics in proteins on a residueby-residue basis [7,8]. We explore the potential for the clustering of molecular dynamics simulations to capture such motions
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