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.

Binding of hepatocyte growth factor (HGF) to the receptor tyrosine kinase MET is implicated in the malignant process of multiple cancers, making disruption of this interaction a promising therapeutic strategy. However, targeting MET with bivalent antibodies can mimic HGF agonism via receptor dimerization. To address this limitation, we have developed onartuzumab, an Escherichia coliderived, humanized, and affinity-matured monovalent monoclonal antibody against MET, generated using the knob-into-hole technology that enables the antibody to engage the receptor in a one-to-one fashion. Onartuzumab potently inhibits HGF binding and receptor phosphorylation and signaling and has antibody-like pharmacokinetics and antitumor activity. Biochemical data and a crystal structure of a ternary complex of onartuzumab antigen-binding fragment bound to a MET extracellular domain fragment, consisting of the MET Sema domain fused to the adjacent Plexins, Semaphorins, Integrins domain (MET Sema-PSI), and the HGF β-chain demonstrate that onartuzumab acts specifically by blocking HGF α-chain (but not β-chain) binding to MET. These data suggest a likely binding site of the HGF α-chain on MET, which when dimerized leads to MET signaling. Onartuzumab, therefore, represents the founding member of a class of therapeutic monovalent antibodies that overcomes limitations of antibody bivalency for targets impacted by antibody crosslinking.scatter factor | HGFR | MetMAb | OA5D5

Lacking any discernible sequence similarity, interleukin-34 (IL-34) and colony stimulating factor 1 (CSF-1) signal through a common receptor CSF-1R on cells of mononuclear phagocyte lineage. Here, the crystal structure of dimeric IL-34 reveals a helical cytokine fold homologous to CSF-1, and we further show that the complex architecture of IL-34 bound to the N-terminal immunoglobulin domains of CSF-1R is similar to the CSF-1/CSF-1R assembly. However, unique conformational adaptations in the receptor domain geometry and intermolecular interface explain the cross-reactivity of CSF-1R for two such distantly related ligands. The docking adaptations of the IL-34 and CSF-1 quaternary complexes, when compared to the stem cell factor assembly, draw a common evolutionary theme for transmembrane signaling. In addition, the structure of IL-34 engaged by a Fab fragment reveals the mechanism of a neutralizing antibody that can help deconvolute IL-34 from CSF-1 biology, with implications for therapeutic intervention in diseases with myeloid pathogenic mechanisms.

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 ␣F helix away from the heme pocket, as this helix is normally held in place via the inhibitory His 105 -heme covalent bond. The structure provides insights into how BAY 58 -2667 binds and activates sGC to rescue heme-NO dysfunction in cardiovascular diseases.

The molecular mechanism of desensitization of soluble guanylyl cyclase (sGC), the NO receptor, has long remained unresolved. Posttranslational modification and redox state have been postulated to affect sGC sensitivity to NO but evidence has been lacking. We now show that sGC can be S-nitrosylated in primary aortic smooth muscle cells by S-nitrosocysteine (CSNO), an S-nitrosylating agent, in human umbilical vein endothelial cells after vascular endothelial growth factor treatment and in isolated aorta after sustained exposure to acetylcholine. Importantly, we show that S-nitrosylation of sGC results in decreased responsiveness to NO characterized by loss of NO-stimulated sGC activity. Desensitization of sGC is concentration-and time-dependent on exposure to CSNO, and sensitivity of sGC to NO can be restored and its S-nitrosylation prevented with cellular increase of thiols. We confirm in vitro with semipurified sGC that S-nitrosylation directly causes desensitization, suggesting that other cellular factors are not required. Two potential S-nitrosylated cysteines in the ␣-and ␤-subunits of sGC were identified by MS. Replacement of these cysteines, C243 in ␣ and C122 in ␤, created mutants that were mostly resistant to desensitization. Structural analysis of the region near ␤-C122 in the homologous Nostoc H-NOX crystal structure indicates that this residue is in the vicinity of the heme and its S-nitrosylation could dampen NO activation by affecting the positions of key residues interacting with the heme. This study suggests that S-nitrosylation of sGC is a means by which memory of NO exposure is kept in smooth muscle cells and could be a mechanism of NO tolerance.cGMP ͉ tolerance ͉ redox ͉ S-nitrosothiols I n the cardiovascular system, nitric oxide (NO) is critical for regulation of vascular tone and homeostasis (1, 2). In mammals, the main sensor of NO is the soluble guanylyl cyclase (sGC), a heme-containing heterodimer formed by an ␣-and a ␤-subunit. When NO binds to the heme, catalytic activity of sGC increases several hundredfold to produce the second messenger cGMP (3). Despite the importance of the NO-cGMP pathway in the biology and pathology of the cardiovascular and neuronal systems, modulation of sGC is poorly understood (4, 5). In particular, the mechanism of desensitization of sGC has remained unresolved despite 30 years of effort. Desensitization is the transition to a state in which sGC's response to a new NO stimulation is reduced or abolished. This direct effect on sGC differs from the desensitization of the NO-cGMP pathway caused by decrease in cGMP levels [e.g., because of increased phosphodiesterase activity (6)] or decrease in NO availability (7). Desensitization of sGC itself has been reported in various cell types and tissues after exposure to NO (8-11) but its mechanism is unknown. Ser/Thr phosphorylation was proposed to be involved but evidence is still lacking (12), and Tyr phosphorylation was ruled out recently (13). Elucidating the mechanism of sGC desensitization is crucial considering i...