Soluble guanylyl/guanylate cyclase (sGC) converts GTP to cGMP after binding nitric oxide, leading to smooth muscle relaxation and vasodilation. Impaired sGC activity is common in cardiovascular disease and sGC stimulatory compounds are greatly sought. sGC is a 150 kDa heterodimeric protein with two H-NOX domains (one with heme, one without), two PAS domains, a coiled-coil domain and two cyclase domains. Binding of NO to the sGC heme leads to proximal histidine release and stimulation of catalytic activity. To begin understanding how binding leads to activation, we examined truncated sGC proteins from Manduca sexta (tobacco hornworm) that bind NO, CO and stimulatory compound YC-1, but lack the cyclase domains. We determined the overall shape of truncated Ms sGC using analytical ultracentrifugation and small angle X-ray scattering (SAXS), revealing an elongated molecule 115 Å by 90 Å by 75 Å. Binding of NO, CO or YC-1 had little effect on shape. Using chemical cross-linking and tandem mass spectrometry, we identified 20 intermolecular contacts, allowing us to fit homology models of the individual domains into the SAXS-derived molecular envelope. The resulting model displays a central parallel coiled-coil platform upon which the H-NOX and PAS domains are assembled. The β1 H-NOX and α1 PAS domains are in contact and form the core signaling complex, while the α1 H-NOX domain can be removed without significant effect on ligand binding or overall shape. Removal of 21 residues from the C-terminus yields a protein with dramatically increased proximal histidine release rates upon NO binding.

Soluble guanylyl/guanylate cyclase (sGC), the primary biological receptor for nitric oxide, is required for proper development and health in all animals. We have expressed heterodimeric fulllength and N-terminal fragments of Manduca sexta sGC in Escherichia coli, the first time this has been accomplished for any sGC, and have performed the first functional analyses of an insect sGC. Manduca sGC behaves much like its mammalian counterparts, displaying a 170-fold stimulation by NO and sensitivity to compound YC-1. YC-1 reduces the NO and CO offrates for the ϳ100-kDa N-terminal heterodimeric fragment and increases the CO affinity by ϳ50-fold to 1.7 M. Binding of NO leads to a transient six- Nitric oxide (NO)2 regulates numerous vital functions in animal physiology, including blood pressure, memory formation, platelet aggregation, and tissue development (1). The primary NO receptor is soluble guanylyl/guanylate cyclase (sGC), a heterodimeric protein of ϳ150 kDa that binds NO through a ferrous heme. NO binding stimulates cyclase activity, the production of cGMP from substrate GTP, and the subsequent amplification of NO-dependent signaling cascades (2-5). Although NO is the best described allosteric regulator of sGC, numerous other forms of regulation may also be of importance, including phosphorylation (6), nucleotide binding (7-10), calcium binding (11), nitrosylation (12, 13), and protein-protein interactions (14 -17).The primary form of sGC is an ␣1/␤1 heterodimer composed of two evolutionarily related subunits that display several recognizable domains (Fig. 1A) (18). Heme is bound to the protein through proximal ␤1 His-105. NO binding to the heme leads to proximal histidine release and stimulation of cyclase activity, presumably through a change in protein conformation (reviewed in Ref. 19). The heme-binding domain has evolved from a widespread family of bacterial proteins called H-NOX (Heme-Nitric oxide/OXygen) domain proteins, of which the structures of three are known (20 -22). The central portion of sGC contains two PAS domains (18,23), and the C-terminal region contains a catalytic domain that is very similar to that of adenylyl cyclase (24, 25).In the 1990s, the anti-platelet activity of YC-1, a benzylindazole derivative (Fig. 1B), was found to derive from its ability to bind to and stimulate sGC (26, 27), leading to a search for related compounds that might serve as sGC-targeted drugs for human health (28). The YC-1 mechanism of action remains unclear, as does the location of its binding site in the sGC protein. The nucleotide-like structure of YC-1, in conjunction with mutagenesis studies, has led to the suggestion that YC-1 binds to the cyclase domain (29), whereas cross-linking studies with YC-1-related compounds have indicated that they bind to the N-terminal domain of the ␣1 subunit (30).Studies of insect sGC have lagged behind studies of the mammalian enzyme, but, as in mammals, insect sGC plays an important physiological role. In the Manduca sexta larva (tobacco hornworm), sGC is implicated in ante...

We have determined the 1.65 A crystal structure of human thioredoxin-1 after treatment with S-nitrosoglutathione, providing a high-resolution view of this important protein modification and mechanistic insight into protein transnitrosation. Thioredoxin-1 appears to play an intermediary role in cellular S-nitrosylation and is important in numerous biological and pathobiological activities. S-Nitroso modifications of cysteines 62 and 69 are clearly visible in the structure and display planar cis geometries, whereas cysteines 32, 35, and 73 form intra- and intermolecular disulfide bonds. Surprisingly, the Cys 62 nitroso group is completely buried and pointing to the protein interior yet is the most readily formed at neutral pH. The Cys 69 nitroso group is also protected but requires a higher pH for stable formation. The helix intervening between residues 62 and 69 shifts by approximately 0.5 A to accommodate the SNO groups. The crystallographic asymmetric unit contains three independent molecules of thioredoxin, providing three views of the nitrosated protein. The three molecules are in general agreement but display subtle differences, including both cis and trans conformers for Cys 69 SNO in molecule C, and greater disorder in the Cys 62-Cys 69 helix in molecule B. Possible mechanisms for protein transnitrosation with specific geometric requirements and charge stabilization of the nitroxyl disulfide reaction intermediate are discussed.

Oxidation and loss of heme in soluble guanylyl/guanylate cyclase (sGC), the nitric oxide receptor, is thought to be a major contributor to cardiovascular disease and is the target of compounds BAY 58-2667 and HMR1766. Using spectroelectrochemical titration, we found a truncated sGC to be highly stable in the ferrous state (+234 mV) and to bind ferrous heme tightly even in the presence of NO, despite the NO-induced release of the proximal histidine. In contrast, oxidized sGC readily loses ferric heme to myoglobin (0.47 ± 0.02 hr−1). Peroxynitrite, the presumed cellular oxidant, readily oxidizes sGC in 5 mM glutathione.

The objectives of this study were to quantity and compare the activities of a minimal heat shock (HS) promoter and other promoters used in gene therapy applications, and to identify strategies to amplify the heat inducibility of therapeutic genes. Human tumour cells were transiently or stably transfected with the HS promoter driving expression of reporter genes. HS promoter activity was induced transiently, with maximum activity 16-24 h after HS, and was dependent on temperature. The activity of the minimal HS promoter was similar, after 42 degrees C HS for 1 h, to that of the cytomegalovirus (CMV) promoter. To determine if the HS promoter could be used to activate a second conditional promoter, cells were transiently transfected with vectors containing both the HS and human immunodeficiency virus type 1 (HIV1) promoters. When the IL-2 gene was placed downstream of the HIV1 promoter. IL-2 production was temperature-independent. The addition of the HIV tat gene downstream of the HS promoter caused IL-2 to be induced more than 3 fold after a single 42 degrees C HS. These data indicate that the minimal HS promoter, following activation by clinically attainable temperatures (< or = 42 degrees C), can drive expression of therapeutic genes at levels comparable to the CMV promoter and be used in conjunction with a second conditional promoter to drive temperature-dependent, gene expression.