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Heme-based NO sensors HNOX: Heme-nitric oxide/oxygen binding domain/protein Bacterial 1 J. Inorg. Biochem. 99, 892 (2005). Soluble guanylate cyclase (sGC) is a nitric oxide (NO) sensing hemoprotein that has been found in eukaryotes from Drosophila to humans. Prokaryotic proteins with significant homology to the heme domain of sGC have recently been identified through genomic analysis. This family of heme proteins has been named the H-NOX domain, for Heme-Nitric oxide/OXygen binding domain. The key observation from initial studies in this family is that some members, those proteins 2 3 [B] sensor protein - HnoX (4) HnoX N N Fe N N SIGNAL NO dissociation HnoC, HnoD or HnoB response regulators histidine kinase HnoK Histidine kinase Asp His ATP P autophosphorylation P phosphotransfer LuxU HqsK Histidine kinase Asp ATP P His ATP autophosphorylation regulation of transcription or catalytic activity LuxO Histidine kinase His P OUPUT P Asp P OUTPUT regulation of transcription phosphotransfer Fig. 4 4 Sessility (biofilm formation) Fimbrial formation Virulence Environmental persistence Cell-cell communication INPUT (signal) Binding of signal molecules Phosphorylation Ion binding DNA binding Light O N OH O HO P O N N HN OUTPUT (phenotype) NH2 O O N N O O H2N NH O P OH O HO N Phosphodiesterase c-di GMP O O N O P O O Diguanylate cyclase NH OH N N NH2 HO HO OH N H2N HN N O OH O O P O N O HO HO N pGpG or l-di GMP OUTPUT (phenotype) O Motility Virulence Phage resistance Hyphae formation Antibiotic production OH OH O P P O O P O N HO HO OH OH O HO O N HN N N O OH O O H2N NH O N O Fig. 7 P HO GTP O O P O P OH NH2 INPUT (signal) O HO Binding of signal molecules Phosphorylation Ion binding DNA binding 5 Light O LuxN CqsS N LuxPQ LuxO Asp P P His P His Asp Asp LuxU P His His P Asp Hpt Fur His P HnoK HqsK Asp P His His Asp P HnoC HnoX HqsK Asp ATP ATP Fe His P ADP ADP histidine kinase DGC ATP ADP His P response regulator Asp P ? His P HnoB Asp P PE transcriptional feedback Asp P HnoK PE HnoD 2 GTP c-diGMP l-diGMP Fig. 10 Asp P DGC 6 The Marletta Lab. Univ. California, Berkeley, Script Research Institute Genomic analysis has recently placed sGC within a larger family of proteins with Heme Nitric oxide/Oxygen binding (H-NOX) domains including prokaryotic proteins with significant homology (1540% identity) to the heme domain of sGC. Predicted H-NOX domains were found in facultative aerobes, obligate anaerobes, and thermophiles. Genomic analysis reveals that the H-NOX domains may be linked to histidine kinases or diguanylate cyclases (obligate anaerobes) or methyl-accepting chemotaxis proteins (obligate anaerobes). Uncovering the biological function of these H-NOX domains is currently an area of intense investigation in our lab. (A) Structural features of Tt H-NOX. (B) Heme binding pocket.H-NOX proteins also exhibit remarkable diatomic ligand selectivity despite a similar protein fold. For example, the H-NOX domain from Vibrio cholera (a facultative aerobe) binds NO in a high spin 5-coordinate complex and excludes oxygen, while the H-NOX domain from Thermoanaerobacter tengcongensis (Tt, obligate anaerobe) has been found to bind oxygen in a low-spin 6-coordinate complex, making it the first member of the family to bind O2. Current research is focused on understanding the nature of this ligand selectivity from a molecular level and how this selectivity translates into protein function as sensors in biology. 7 Fig. 2. Sequence alignment selected members of the H-NOX family. Sequence numbering is that of Tt H-NOX. Invariant residues are highlighted in green and very highly conserved residues are highlighted in blue. Y140 of Tt H-NOX is highlighted in red. Predicted distal pocket tyrosine residues that may stabilize an FeII–O2 complex in other HNOX proteins are in red. Accession numbers are: Homo sapiens β1 [gi:2746083], Rattus norvegicus β1 [gi:27127318], Drosophila melangaster β1 [gi:861203], Drosophila melangaster CG14885-PA [gi:23171476], Caenorhabditis elegans GCY-35 [gi:52782806], Nostoc punctiforme [gi:23129606], Caulobacter crescentus [gi:16127222], Shewanella oneidensis [gi:24373702], Legionella pneumophila (ORF2) [CUCGC_272624], Clostridium acetobutylicum [gi:15896488], and Thermoanaerobacter tengcongensis [gi:20807169]. Alignments were generated using the program MegAlign. 8 Fig. 3. Speculation on prokaryotic signaling pathways involving H-NOX domains. (a) Proposed role of an H-NOX sensor in a facultative aerobic bacterium. The H-NOX domains in facultative aerobes may have evolved as sensors for NO derived from under conditions of low O2 concentration. The NO signal may be transmitted via the action of a histidine kinase. Most of the predicted H-NOX ORFs from aerobic bacteria are contained within an operons that also contains a predicted histidine kinase, and additionally, these bacteria also contain predicted nitrate reductase proteins, consistent with this hypothesis. (b) Proposed role of an H-NOX sensor in an obligate anaerobic bacterium. The H-NOX domain in obligate anaerobes is fused through a transmembrane domain (shown in gray) to a MCP. Here the H-NOX domain may be used as an O2 sensor to signal a change in O2 concentration, regulating methylation by S-adenosyl-methionine (SAM) leading to taxis towards more favorable O2 concentrations. Aside from NO binding to the H-NOX domain of sGC, ligand binding to an H-NOX protein has not been conclusively linked to biological signaling processes to date. 9 Fig. 4. The heme environment of the Tt H-NOX domain[38]. (a) The conserved Y-S-R motif makes hydrogen bonding interactions with the propionic acid side chains of the heme group, which is colored yellow (porphyrin) and red (iron). (b) The conserved H102 is the proximal ligand to the heme. In Tt H-NOX, a distal pocket hydrogen-bonding network, involving principally Y140, stabilizes an FeII–O2 complex. This hydrogen-bonding network is predicted to be absent in the H-NOX proteins from sGCs and aerobic prokaryotes, suggesting this as a key molecular factor in the remarkable ligand selectivity against O2 displayed by these heme proteins. 10 Fig. 5. Schematic summary of the H-NOX family of heme-based sensors. The progenitor H-NOX domain has evolved to discriminate between ligands such as NO and O2 for specific sensing purposes. This is the first family of related heme proteins to 11 discriminate between different physiologically relevant diatomic gaseous ligands. Mol. Cell 46 (4) 449 (2012) Highlights ► A complex multicomponent bacterial H-NOX signaling network was mapped ► EAL response regulator phosphorylation stimulated c-di-GMP hydrolysis activity ► A degenerate HD-GYP response regulator inhibited the EAL response regulator ► NO induced biofilm formation through c-di-GMP modulation by the signaling network 12 Figure 6. Model of Multicomponent Signaling Network for NO-Induced Biofilm Formation as a Protection Mechanism against NO(A) The complex multicomponent signaling pathway is initiated by NO binding to the sensory H-NOX protein (HnoX), which then inhibits HnoK autophosphorylation. Phosphotransfer establishes a branching of the network to three response regulators. HnoB and HnoD form a feed-forward loop. Phosphorylation controls PDE activity of HnoB, which can be finetuned by allosteric control from HnoD. NO-controlled repression of the PDE activity leads to an increase in c-di-GMP levels, which serves as a signaling cue for cellular attachment into biofilms.(B) The NO signal switches the bacterial motility pattern from planktonic growth to increased attachment onto surfaces. The thick layers of cells provide a protective barrier against diffusion of reactive and damaging NO and may protect cells in the lower 13 layers.