Synergistic stabilisation of NOsGC by cinaciguat and non-hydrolysable nucleotides: Evidence for sGC activator-induced communication between the heme-binding and catalytic domains

Nitric oxide sensitive guanylyl cyclase (NOsGC) is a heterodimeric enzyme consisting of one α and one β subunit. Each subunit consists of four domains: the N-terminal heme-nitric oxide oxygen binding (HNOX) domain, a PAS domain, a coiled-coil domain and the C-terminal catalytic domain. Upon activation by the endogenous ligand NO or activating drugs, NOsGC catalyses the conversion of GTP to cGMP. Although several crystal structures of the isolated domains are known, the structure of the full-length enzyme and the interdomain conformational changes during activation remain unsolved to date. In the current study, we performed protein thermal shift assays of purified NOsGC to identify discrete conformational states amenable to further analysis e.g. by crystallisation. A non-hydrolysable substrate analogue binding to the catalytic domain led to a subtle change in melting temperature. An activator drug binding to the HNOX domain led to a small increase. However, the combination of substrate analogue and activator drug led to a marked synergistic increase from 51 °C to 60 °C. This suggests reciprocal communication between HNOX domain and catalytic domain and formation of a stable activated conformation amenable to further biophysical characterization.