P64: Biochemical and structural characterization of the soluble guanylate cyclase catalytic domain

• Published in: Nitric oxide Vol. 31; p. S40
• Main Authors: Garcin, Elsa D., Seeger, Franziska
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 15.04.2013
• ISSN: 1089-8603; 1089-8611
• DOI: 10.1016/j.niox.2013.02.066

Soluble guanylate cyclase (sGC) is a key enzyme in the NO–sGC–cGMP signaling cascade and is crucial to cardiovascular system regulation. NO binding to the 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 the NO–sGC–cGMP system results in hypertension and acute heart failure. Nitrite delivery has been shown to enhance hypoxic NO signaling via cGMP production. sGC is a heterodimer of two homologous subunits, α and β, which both contain three domains: an N-terminal regulatory domain (HNOX: Heme Nitric oxide OXygen), a central dimerization HNOX associated (HNOXA) and coiled-coil (CC) domain, and a C-terminal catalytic domain (GC). The sGC 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. 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. Here, we report the first crystal structure of wild type human heterodimeric αβGC at 1.9Å resolution as well as activity data comparing full-length sGC to our truncated catalytic constructs. The cGMP activity measurements show that αβCC–GC exhibits higher levels of catalytic activity than both abGC and basally active full-length sGC. Activity levels of αβCC–GC compared to basal and activated full-length sGC and abGC hint at structural differences between the two constructs and full-length sGC. Our results also suggest that additional sGC domains are necessary for full enzymatic activity. These differences will be further characterized by structural studies using protein X-ray crystallography. Supported by American Heart Association Scientist Development Grant.