Molecular dynamics-based identification of binding pathways and two distinct high-affinity sites for succinate in succinate receptor 1/GPR91.

SUCNR1 is an auto- and paracrine sensor of the metabolic stress signal succinate. Using unsupervised molecular dynamics (MD) simulations (170.400 ns) and mutagenesis across human, mouse, and rat SUCNR1, we characterize how a five-arginine motif around the extracellular pole of TM-VI determines the initial capture of succinate in the extracellular vestibule (ECV) to either stay or move down to the orthosteric site. Metadynamics demonstrate low-energy succinate binding in both sites, with an energy barrier corresponding to an intermediate stage during which succinate, with an associated water cluster, unlocks the hydrogen-bond-stabilized conformationally constrained extracellular loop (ECL)-2b. Importantly, simultaneous binding of two succinate molecules through either a "sequential" or "bypassing" mode is a frequent endpoint. The mono-carboxylate NF-56-EJ40 antagonist enters SUCNR1 between TM-I and -II and does not unlock ECL-2b. It is proposed that occupancy of both high-affinity sites is required for selective activation of SUCNR1 by high local succinate concentrations. [Display omitted] • SUCNR1 attracts and binds succinate through a cluster of arginines around TM-VI • The arginines constitute two low-energy succinate binding sites • SUCNR1 is activated by simultaneous binding of two succinates at high concentrations • Binding of succinate, but not antagonist, unlocks constrained ECL-2 via water cluster Extracellular succinate is an auto- and paracrine metabolic stress signal sensed by SUCNR1. Using MD simulations, Shenol et al. found that SUCNR1 has two low-energy binding sites, and its selective activation by high, local concentrations of succinate requires simultaneous occupancy of both high-affinity sites. [ABSTRACT FROM AUTHOR]