Nitric oxide-sensing H-NOX proteins govern bacterial communal behavior

Abstract

Heme-nitric oxide/oxygen binding (H-NOX) domains function as sensors for the gaseous signaling agent nitric oxide (NO) in eukaryotes and bacteria. Mammalian NO signaling is well characterized and involves the H-NOX domain of soluble guanylate cyclase. In bacteria, H-NOX proteins interact with bacterial signaling proteins in two-component signaling systems or in cyclic-di-GMP metabolism. Characterization of several downstream signaling processes has shown that bacterial H-NOX proteins share a common role in controlling important bacterial communal behaviors in response to NO. The H-NOX pathways regulate motility, biofilm formation, quorum sensing, and symbiosis. Here, we review the latest structural and mechanistic studies that have elucidated how H-NOX domains selectively bind NO and transduce ligand binding into conformational changes that modulate activity of signaling partners. Furthermore, we summarize the recent advances in understanding the physiological function and biochemical details of the H-NOX signaling pathways.

Keywords: H-NOX; biofilm; cyclic-di-GMP; nitric oxide; quorum sensing; two-component signaling.

Fig. 1. X-ray crystal structure of Thermoanaerobacter tengcongensis (Tt) H-NOX

A. Structure of the H-NOX domain showing the N-terminal subdomain on top and C-terminal subdomain on the bottom with the heme cofactor buried in a pocket. The heme-coordinating H102 residue and α-helix F are at the bottom (light red). B. Close-up of the heme-binding pocket. The YxSxR residues are shown in orange.

Fig. 2. Distribution of selected bacterial H-NOX genes and operon organization

A. Phylogenetic tree of select bacterial species containing H-NOX genes. To the right, the gene organization of the H-NOX containing operons is schematized. Operons shaded in yellow contain predicted O₂-binding H-NOX proteins based on the presence of a Tyr residue in the distal pocket. The figure was generated using the Interactive Tree of Life [80]. B. Euler diagram displaying the number of H-NOX containing species that possess each of the listed downstream effector genes in the H-NOX operon.

Fig. 3. Determinants of ligand selectivity for O₂ and NO binding

A. The heme pocket of the O₂-binding H-NOX protein from T. tengcongensis (PDB: 1U55) containing the H-bonding network consisting of Y140, W9, N74. B. The hydrophobic heme-pocket of the NO-selective H-NOX protein from Nostoc sp. (PDB: 2O09). C. Additional factors involved in tuning the ligand binding affinity of H-NOX proteins include heme distortion, distal pocket bulk and protein flexibility, as well as ligand access through tunnels into the heme pocket.

Fig. 4. Ligand-induced activation mechanism

A. Structural alignment of selected Tt H-NOX crystal structures (left) displaying the rotational displacement of the N-terminal subdomain with respect to the C-terminal subdomain. Alignment of the heme cofactors on the right displays the varying degree of heme distortion associated with each protein conformation. B. Heme-strain model for H-NOX activation. In the unliganded H-NOX state, the heme is highly distorted due to van-der-Waals interaction between P115 and I5 (Tt H-NOX numbering) with two of the heme pyrroles. Initial formation of a six-coordinate Fe(II)-NO complex weakens the iron-His bond leading to His dissociation. Formation of the 5-coordinate Fe(II)-NO complex allows relaxation of the heme into a more planar geometry. Contacts between the N-terminal helix and the heme, in particular through I5, trigger an upward rotational displacement of the N-terminal subdomain (top) relative to the C-terminal subdomain (bottom).

Fig. 5. H-NOX-dependent control of biofilm formation through regulation of cyclic-di-GMP levels

A. H-NOX signaling in L. pneumophila and S. woodyi. The H-NOX protein HnoX interacts with a dual diguanylate cyclase/phosphodiesterase (DGC/PDE) enzyme. The NO-bound H-NOX state inhibits DGC activity, and in the case of S. woodyi, also activates PDE activity, leading to increased c-di-GMP hydrolysis and lower biofilm formation in reponse to NO. B. H-NOX signaling promotes biofilm formation in response to NO in S. oneidensis and V. cholerae. The NO-bound H-NOX state inhibits autophosphorylation of the associated histidine kinase HnoK. HnoK possesses three phosphotransfer targets: HnoB, HnoC, and HnoD. HnoB contains a phosphorylation-activated PDE domain that hydrolyzes c-di-GMP. HnoD functions as a phosphorylation-dependent allosteric inhibitor of HnoB to fine-tune c-di-GMP hydrolysis. HnoC serves as a transcriptional regulator of the signaling genes in the network for further feedback control.

Fig. 6. H-NOX-dependent control of host colonization in V. fischeri and cross-talk with quorum sensing in V. harveyi

A. H-NOX signaling in V. fischeri in the symbiosis with the Hawaiian bobtail squid E. scolopes. Bacteria encounter NO during colonization of the squid light organ. NO-bound H-NOX inhibits autophosphorylation of the associated HK. Through an uncharacterized phosphorelay system, the NO-bound HnoX downregulates the expression of a gene set containing a Fur-binding motif in the promoter region. A subset of genes repress hemin-specific iron-uptake and utilization genes. Downregulation of intracellular iron levels in response to NO primes V. fischeri for exposure to ROS during the course of colonization. B. Interaction between the H-NOX signaling and quorum sensing pathway in V. harveyi. Quorum sensing autoinducers control the activity of three sensor HKs (CqsS, LuxN and LuxPQ). Phosphotransfer from all three HKs to a single Hpt protein, LuxU, and a RR, LuxO, integrates the quorum sensing signal. LuxO controls the expression of small regulatory RNAs Qrr1–5, which in turn repress LuxR expression. LuxU is the final transcriptional activator of quorum sensing genes, regulating bioluminescence and other communal responses. NO-bound HnoK inhibits the activity of the associated HqsK histine kinase. HqsK is capable of also phosphorylating LuxU, linking NO sensing to expression of quorum sensing genes.