Interfacial Residues Promote an Optimal Alignment of the Catalytic Center in Human Soluble Guanylate Cyclase: Heterodimerization Is Required but Not Sufficient for Activity

Abstract

Soluble guanylate cyclase (sGC) plays a central role in the cardiovascular system and is a drug target for the treatment of pulmonary hypertension. While the three-dimensional structure of sGC is unknown, studies suggest that binding of the regulatory domain to the catalytic domain maintains sGC in an autoinhibited basal state. The activation signal, binding of NO to heme, is thought to be transmitted via the regulatory and dimerization domains to the cyclase domain and unleashes the full catalytic potential of sGC. Consequently, isolated catalytic domains should show catalytic turnover comparable to that of activated sGC. Using X-ray crystallography, activity measurements, and native mass spectrometry, we show unambiguously that human isolated catalytic domains are much less active than basal sGC, while still forming heterodimers. We identified key structural elements regulating the dimer interface and propose a novel role for residues located in an interfacial flap and a hydrogen bond network as key modulators of the orientation of the catalytic subunits. We demonstrate that even in the absence of the regulatory domain, additional sGC domains are required to guide the appropriate conformation of the catalytic subunits associated with high activity. Our data support a novel regulatory mechanism whereby sGC activity is tuned by distinct domain interactions that either promote or inhibit catalytic activity. These results further our understanding of heterodimerization and activation of sGC and open additional drug discovery routes for targeting the NO–sGC–cGMP pathway via the design of small molecules that promote a productive conformation of the catalytic subunits or disrupt inhibitory domain interactions.

International Union of Basic and Clinical Pharmacology. CI. Structures and Small Molecule Modulators of Mammalian Adenylyl Cyclases

“…At present, the PDB provides 28 sAC crystal structures (Kleinboelting et al, 2014a(Kleinboelting et al, ,b, 2016Saalau-Bethell et al, 2014;RamosEspiritu et al, 2016), including complexes with ATP, a,b-methylene adenosine-59-triphosphate, cAMP, pyrophosphate, bicarbonate, biselenite, bisulfite, bithionol, and various inhibitors. Comparing structures of 5C1: 2C2, of sAC-C1-C2 and of an inactive human sGCa cat : sGCb cat heterodimer (Allerston et al, 2013;Seeger et al, 2014) indicates high structural homology not only of the individual catalytic domains, but also of the whole complexes, and points to a common, well conserved mechanism of cNMP generation facilitated by two metal ions.…”

The Influence of Nitric Oxide on Soluble Guanylate Cyclase Regulation by Nucleotides

“…Crystal structures of the sGC catalytic domain (Protein Data Bank entries 3UVJ, 2WZ1, and 4NI2) suggest two binding sites for nucleotides located in the interface between the two domains (23,24). One of these sites is the active site, and the other is the pseudosymmetric site.…”

“…To date, all reported crystal structures of the sGC catalytic domain are in a putative inactive (open) conformation and lack bound nucleotides (Protein Data Bank entries 3UVJ, 2WZ1, and 4NI2) (23,24). Modeling the active (closed) conformation of sGC based on AC structures also reveals a pseudosymmetric cavity (Fig.…”

Structure/function of the soluble guanylyl cyclase catalytic domain

“…Several structures of apo inactive cyclase domains from bacteria, algae, fungus, and human have been solved, but a structure of the holo active form remains elusive [40–44]. Several groups have reported a high propensity for ββGC cat homodimers to form both in solution and during crystallization attempts [42,43]. Despite these structural characterizations, several key questions remain about the αβGC cat domain: what is the mechanism by which αβGC cat transitions from the inactive apo conformation to the catalytically-active conformation upon NO binding?…”

“…3g and h) in its apo inactive state [43]. Comparison with the mutant heterodimer structure [42] reveals how subtle conformational changes at the dimer interface can affect catalytic activity.…”