The gasotransmitter nitric oxide (NO) is a critical endogenous regulator
of homeostasis, in major part via the generation of cGMP (cyclic
guanosine monophosphate) from GTP (guanosine triphosphate) by NO’s main
physiological receptor, the soluble guanylate cyclase (sGC). sGC is a
heterodimer, composed of an alpha 1 and a beta 1 subunit, of which the
latter contains the heme-nitric oxide/oxygen (H-NOX) domain, responsible
for NO recognition, binding and signal initiation. The NO/sGC/cGMP axis
is dysfunctional in a variety of diseases, including hypertension and
heart failure, especially since oxidative stress results in heme
oxidation, sGC unresponsiveness to NO and subsequent degradation. As a
central player in this axis, sGC is the focus of intense research
efforts aiming to develop therapeutic molecules that enhance its
activity. A class of drugs named sGC “activators” aim to replace the
oxidized heme of the H-NOX domain, thus stabilizing the enzyme and
restoring its activity. Although numerous studies outline the
pharmacology and binding behavior of these compounds, the static 3D
models available so far do not allow a satisfactory understanding of the
structural basis of sGC’s activation mechanism by these drugs. Herein,
application NMR describes different conformational states during the
replacement of the heme by a sGC activators. We show that the two sGC
activators (BAY 58-2667 and BAY 60-2770) significantly decrease the
conformational plasticity of the recombinant H-NOX protein domain of
Nostoc sp. cyanobacterium, rendering it a lot more rigid compared to the
heme-occupied H-NOX. NMR methodology also reveals, for the first time, a
surprising bi-directional competition between reduced heme and these
compounds, pointing to a highly dynamic regulation of the H-NOX domain.
This competitive, bi-directional mode of interaction is also confirmed
by monitoring cGMP generation in A7r5 vascular smooth muscle cells by
these activators. We show that, surprisingly, heme’s redox state impacts
differently the bioactivity of these two structurally similar compounds.
In all, by NMR-based and functional approaches we contribute unique
experimental insight into the dynamic interaction of sGC activators with
the H-NOX domain and its dependence on the heme redox status, with the
ultimate goal to permit a better design of such therapeutically
important molecules