(192z) Capturing Differences in Dynamics of Structurally Similar Signaling Proteins

Although many homologous proteins have highly conserved structural motifs as well as overall structural folds, the functional characteristics of such proteins are strikingly different in solution, indicating a role of dynamic motions. The differences in dynamics of closely-related proteins present significant hurdles for structure-based design of small molecules targeting such proteins. In this work, we have studied the interplay of protein dynamics and small-molecule inhibition in a set of signaling proteins, known as Regulators of G protein signaling (RGS) proteins, that modulate signaling in G-protein coupled receptors by binding to Gα-subunits of heterotrimeric G proteins and accelerating hydrolysis of GTP. While structures of several known RGS proteins largely contain α-helical motifs, some thiadiazolidinone (TDZD) compounds that target cysteine residues have shown different levels of specificities and potencies for closely related RGS proteins, thereby indicating intrinsic differences in their dynamics. We have characterized such differences in three different RGS proteins (apo-RGS4, apo-RGS8, and apo-RGS19) using microsecond-scale classical molecular dynamics (MD) simulations with CHARMM and AMBER force-fields. Analyses of these trajectories reveal high fluctuations in α5 and α6 helices and in the loops connecting them that facilitate exposure of deeply buried cysteine residues to allosteric inhibitors. We further validate our findings using hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis of all three proteins and provide details on exchange rates for hydrogen atoms at the resolution of single residues.