Biochemical and structural characterization of the activation of soluble Guanylate Cyclase

Abstract

Soluble Guanylate Cyclase (sGC) is a heterodimeric enzyme converting GTP to cyclic GMP. Each subunit contains an N‐terminal, heme‐containing regulatory domain (HNOX: Heme Nitric oxide OXygen), a dimerization PAS (Per/Arnt/Sim) and coiled‐coil (CC) domain, and a C‐terminal catalytic domain (GC). Controlling crucial physiological functions such as vasorelaxation, a low turnover of sGC causes cardiovascular pathologies. Binding of nitric oxide (NO) to HNOX maximally activates sGC and alleviates a presumed inhibitory effect of HNOX on GC. However, the isolated GC exhibits low activity excluding HNOX as the only brake on catalytic turnover.To elucidate the activation mechanism and identify the domains required for maximum activity, we are studying truncated bovine sGC constructs. These include GC, CC‐GC, and PAS‐CC‐GC as well as the HNOX‐PAS and HNOX‐PAS‐CC heterodimers.We report preliminary SAXS (Small Angle X‐ray Scattering) ab initio envelopes on HNOX‐containing constructs and present activity assays for the GC‐containing constructs.Combinations of activity measurements, SAXS, and x‐ray crystallography have been used to elucidate the activation mechanism of sGC. These studies will provide a foundation for structure‐based drug design of novel sGC activators.Work supported by American Heart Association Scientist Development Grant (E. Garcin) and NIH CBI Training Grant NIH T32 GM066706‐06.