MOLECULAR MODEL OF SOLUBLE GUANYLYL CYCLASE: INSIGHT INTO ALLOSTERY IN NITRIC OXIDE SIGNALING

Abstract

Soluble guanylyl cyclase (sGC), the nitric oxide (NO) receptor, is a 150 kDa heterodimeric multi-domain protein that contains heme in the β subunit. Binding of NO to heme leads to rupture of the proximal histidine bond, increased catalytic conversion of GTP to cGMP at a distant guanylyl cyclase catalytic domain, and vasodilation through cGMP signaling. The structure of sGC has not been determined, and little is known about the mechanism by which NO binding to heme leads to increased catalysis. The small molecule YC-1 is known to stimulate sGC activity, but the exact YC-1 binding site and mechanism of action are unknown. Using truncated constructs of Manduca sexta (Ms) sGC lacking the catalytic domain, conformational changes upon YC-1 and NO-binding were characterized using analytical ultracentrifugation and small-angle X-ray scattering. Chemical cross-linking and high-resolution mass spectrometry was used to obtain distance restraints which, when combined with homology models, have provided the first model of sGC domain arrangement and revealed important information about domain-domain interactions. Truncated Ms sGC is highly elongated, contains a coiled-coil in a parallel arrangement, and contains a direct interface between the β H-NOX (Heme Nitric oxide/Oxygen binding domain) and the coiled-coil, and between the β H-NOX and α PAS (Per-arnt-sim) domains. Experiments using analytical ultracentrifugation, fluorescence anisotropy and native mass spectrometry have revealed the YC-1 binding site to be located within the α PAS domain. Additionally, measurement of the kinetics of heme loss and the heme reduction potential were performed to investigate the instability of oxidized sGC heme.