Diatomic ligand discrimination by soluble guanylyl cyclase (sGC) is paramount to cardiovascular homeostasis and neuronal signaling. Nitric oxide (NO) stimulates sGC activity 200‐fold compared with only four‐fold by carbon monoxide (CO). The molecular details of ligand discrimination and differential response to NO and CO are not well understood. These ligands are sensed by the heme domain of sGC, which belongs to the heme nitric oxide oxygen (H‐NOX) domain family, also evolutionarily conserved in prokaryotes. Here we report crystal structures of the free, NO‐bound, and CO‐bound H‐NOX domains of a cyanobacterial homolog. These structures and complementary mutational analysis in sGC reveal a molecular ruler mechanism that allows sGC to favor NO over CO while excluding oxygen, concomitant to signaling that exploits differential heme pivoting and heme bending. The heme thereby serves as a flexing wedge, allowing the N‐terminal subdomain of H‐NOX to shift concurrent with the transition of the six‐ to five‐coordinated NO‐bound state upon sGC activation. This transition can be modulated by mutations at sGC residues 74 and 145 and corresponding residues in the cyanobacterial H‐NOX homolog.

The number and diversity of β-lactamases are steadily increasing. The emergence of β-lactamases that hydrolyze carbapenems poses a significant threat to our antibiotic armamentarium. The explosion of OXA enzymes that are carbapenem hydrolyzers is a major challenge (carbapenem-hydrolyzing class D [CHD]). An urgent need exists to discover β-lactamase inhibitors with class D activity. The sulbactam-ETX2514 combination demonstrates the potential to become a treatment regimen of choice for Acinetobacter spp. producing class D β-lactamases.

Heme is a vital molecule for all life forms with heme being capable of assisting in catalysis, binding ligands, and undergoing redox changes. Heme-related dysfunction can lead to cardiovascular diseases with the oxidation of the heme of soluble guanylyl cyclase (sGC) critically implicated in some of these cardiovascular diseases. sGC, the main nitric oxide (NO) receptor, stimulates second messenger cGMP production, whereas reactive oxygen species are known to scavenge NO and oxidize/inactivate the heme leading to sGC degradation. This vulnerability of NO-heme signaling to oxidative stress led to the discovery of an NO-independent activator of sGC, cinaciguat (BAY 58–2667), which is a candidate drug in clinical trials to treat acute decompensated heart failure. Here, we present crystallographic and mutagenesis data that reveal the mode of action of BAY 58–2667. The 2.3-Å resolution structure of BAY 58–2667 bound to a heme NO and oxygen binding domain (H-NOX) from Nostoc homologous to that of sGC reveals that the trifurcated BAY 58–2667 molecule has displaced the heme and acts as a heme mimetic. Carboxylate groups of BAY 58–2667 make interactions similar to the heme-propionate groups, whereas its hydrophobic phenyl ring linker folds up within the heme cavity in a planar-like fashion. BAY 58–2667 binding causes a rotation of the αF helix away from the heme pocket, as this helix is normally held in place via the inhibitory His¹⁰⁵–heme covalent bond. The structure provides insights into how BAY 58–2667 binds and activates sGC to rescue heme-NO dysfunction in cardiovascular diseases.

β-Lactamase inhibition is an important clinical strategy in overcoming β-lactamase-mediated resistance to β-lactam antibiotics in Gram negative bacteria. A new β-lactamase inhibitor, avibactam, is entering the clinical arena and promising to be a major step forward in our antibiotic armamentarium. Avibactam has remarkable broad-spectrum activity in being able to inhibit classes A, C, and some class D β-lactamases. We present here structural investigations into class A β-lactamase inhibition by avibactam as we report the crystal structures of SHV-1, the chromosomal penicillinase of Klebsiella pneumoniae, and KPC-2, an acquired carbapenemase found in the same pathogen, complexed with avibactam. The 1.80 Å KPC-2 and 1.42 Å resolution SHV-1 β-lactamase avibactam complex structures reveal avibactam covalently bonded to the catalytic S70 residue. Analysis of the interactions and chair-shaped conformation of avibactam bound to the active sites of KPC-2 and SHV-1 provides structural insights into recently laboratory-generated amino acid substitutions that result in resistance to avibactam in KPC-2 and SHV-1. Furthermore, we observed several important differences in the interactions with amino acid residues, in particular that avibactam forms hydrogen bonds to S130 in KPC-2 but not in SHV-1, that can possibly explain some of the different kinetic constants of inhibition. Our observations provide a possible reason for the ability of KPC-2 β-lactamase to slowly desulfate avibactam with a potential role for the stereochemistry around the N1 atom of avibactam and/or the presence of an active site water molecule that could aid in avibactam desulfation, an unexpected consequence of novel inhibition chemistry.