BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF CATALYTIC AND REGULATORY CONSTRUCTS OF SOLUBLE GUANYLATE CYCLASE

Soluble guanylate cyclase (sGC) is a key enzyme in the NO-sGC-cGMP signaling cascade and is crucial to cardiovascular regulation. NO binding to sGC regulatory domain enhances its basal catalytic activity to convert GTP to cGMP. The second messenger cGMP modulates downstream targets leading to vasodilation. Low output of this system results in hypertension and acute heart failure, two leading causes of death globally. sGC is a heterodimer of two homologous subunits: α and β. Each subunit contains an N-terminal regulatory domain (HNOX: Heme Nitric oxide OXygen), central dimerization HNOX associated (HNOXA) and coiled coil (CC) domains, and a C-terminal catalytic domain (GC). The enzyme is basally active in the absence of NO, but NO binding to the heme group of the β subunit HNOX domain enhances catalytic output several hundred fold. This domain acts as a brake on catalytic activity, but the mechanism by which the HNOX domain inhibits activity in the basal state and relays the NO activation signal to the catalytic domain remains elusive. By studying truncated sGC catalytic constructs, abGC and abCC-GC, we determined structural features necessary for enzymatic activity. Here, we report the first 1.9 � crystal structure of wild type human heterodimeric αβGC and specific catalytic activity normalized per molecule of αβGC heterodimer using native mass spectrometry. cGMP activity measurements show that the isolated catalytic domain only exhibits 0.01% of the basal activity of full-length sGC despite its ability to heterodimerize. We propose that additional domains are needed to achieve sGC full catalytic output. We further provide preliminary data suggesting that 29 C-terminal residues in αGC regulate enzymatic activity by modulating the heterodimeric interface of sGC catalytic domains. Preliminary small-angle X-ray scattering and computational modeling data provide first evidence for spatial domain orientations of the HNOX and HNOXA domains in heme-free homodimeric MBP-βHNOX-HNOXA-CCtruncated construct. This study provides a knowledge base for individual domain contributions to sGC activation and suggests which conformational changes may occur during activation. As such, the herein reported results could serve as a starting point for the rational structure-aided design of novel sGC activators for the treatment of cardiovascular diseases