Probing the Flexibility of Large Conformational Changes in Protein Structures through Local Perturbations

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Abstract
Protein conformational changes and dynamic behavior are fundamental for such processes as catalysis, regulation, and substrate recognition. Although protein dynamics have been successfully explored in computer simulation, there is an intermediate-scale of motions that has proven difficult to simulate—the motion of individual segments or domains that move independently of the body the protein. Here, we introduce a molecular-dynamics perturbation method, the Rotamerically Induced Perturbation (RIP), which can generate large, coherent motions of structural elements in picoseconds by applying large torsional perturbations to individual sidechains. Despite the large-scale motions, secondary structure elements remain intact without the need for applying backbone positional restraints. Owing to its computational efficiency, RIP can be applied to every residue in a protein, producing a global map of deformability. This map is remarkably sparse, with the dominant sites of deformation generally found on the protein surface. The global map can be used to identify loops and helices that are less tightly bound to the protein and thus are likely sites of dynamic modulation that may have important functional consequences. Additionally, they identify individual residues that have the potential to drive large-scale coherent conformational change. Applying RIP to two well-studied proteins, Dihdydrofolate Reductase and Triosephosphate Isomerase, which possess functionally-relevant mobile loops that fluctuate on the microsecond/millisecond timescale, the RIP deformation map identifies and recapitulates the flexibility of these elements. In contrast, the RIP deformation map of α-lytic protease, a kinetically stable protein, results in a map with no significant deformations. In the N-terminal domain of HSP90, the RIP deformation map clearly identifies the ligand-binding lid as a highly flexible region capable of large conformational changes. In the Estrogen Receptor ligand-binding domain, the RIP deformation map is quite sparse except for one large conformational change involving Helix-12, which is the structural element that allosterically links ligand binding to receptor activation. RIP analysis has the potential to discover sites of functional conformational changes and the linchpin residues critical in determining these conformational states. Many proteins undergo large motions to carry out their biological functions. The exact nature of these motions is typically inferred from the crystal structures of the protein trapped in different states, which normally constitutes a difficult series of experiments. As molecular dynamics is generally accepted to accurately model the motion of proteins, the promise is that a long enough simulation will generate all the motions of a given protein structure. Unfortunately, current systems run too slowly to simulate all but the smallest motions. To overcome this computational limit, we have developed a molecular-dynamics perturbation method that induces large changes in a protein structure in very short simulation times. The changes correspond to large motions of specific structural elements on the surface of the protein that corroborate well with the canonical motions of several well-characterized proteins. This bodes well for our method to identify, for any given protein structure, structural elements on the surface that might bind drugs, regulate signals, undergo chemical modifications, or become unstructured.