Nitric Oxide Signaling and the H-NOX Domain
The diatomic free radical gas nitric oxide (NO) is an essential component of living systems (4). NO is known to participate in three distinct processes in mammals: vasodilatation, neurotransmission, and innate immunity (Figure 1.1). In the context of vasodilatation, nanomolar concentrations of NO are generated by the endothelium (10), which then acts in a paracrine manner and modulates the tone of blood vessel smooth muscle (12). In the central nervous system (CNS), NO regulates synaptic strength (13). For example, in hippocampal synapses, NO induces long-term potentiation (14-18). In this context, NO may act as retrograde messenger relaying information from the postsynaptic neuron to the presynaptic neuron (19). In the immune system, cells such as macrophages produce micromolar, cytotoxic concentrations of NO in response to pathogen derived molecules (20-24).
Numerous characteristics of NO makes it particularly well suited for intercellular signal transduction (Figure 1.2). NO is small, neutral, and hydrophobic (25, 26), properties that enable it to traverse cell membranes and partition into neighboring cells. Furthermore, the solution decomposition of NO with O2 (mM in the cell) to nitrite and nitrate is second order with respect to NO (22). At the nanomolar concentrations relevant to cell signaling, the rate of the destruction of NO is competitive with diffusion to cellular targets (12). Thus, NO is an ephemeral signal transduction molecule, acting within a limited radius around its site of production. Dynamic signaling events, such as synaptic transmission between neurons in the nervous system, rely on the rapid production and degradation of NO to maintain information flow through physiological systems. Within the cell, NO post-translationally modifies proteins through the process of S-nitrosation, thereby modifying their activity, localization, and interactions (27). S-nitrosation occurs when NO condenses with free thiol moeities such as cysteine residues in proteins (28). Also within the cell, NO is a high affinity ligand for transition metals such as heme iron (29, 30). Heme iron has played a particularly important role in understanding NO biology since its central role in the activation of guanylate cyclase to form cGMP was the first widely recognized NO cell signaling paradigm (31-33).
1.2 The Mammalian NO/cGMP Signal Transduction Pathway: NO Production
The enzyme nitric oxide synthase (NOS) catalyzes the oxidation of the amino acid L-arginine to L-citrulline and NO (Figure 1.3), thereby producing concentrations of NO that participate in cell signal transduction (34). NOS utilizes a P-450-type heme, molecular oxygen (O2), and reducing equivalents provided via the oxidation of NADPH to oxidize arginine to citrulline and NO (35). Three isoforms of nitric oxide synthase (NOS) are present in mammalian genomes: endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS) (35). The three NOS isoforms are multi-component redox enzymes with distinct functional units (Figure 1.4) (35). NOS enzymes possess sequence homology to NADPH cytochrome P-450 reductase and contain cytochrome P-450-type hemes (35). Following reduction and treatment with carbon monoxide (CO), NOS displays a lmax of ~450 nm, typical of P-450 enzymes (35). Despite this property, the catalytic heme domain of NOS lacks any significant sequence homology with the large family of cytochrome P-450 enzymes whose functions are to catalyze oxygenation of endogenous and xenobiotic compounds (35). Thus NOS enzymes lie outside the P-450 superfamily, but utilize a P-450-type heme to perform chemistry. All three NOS variants possess a regulatory calmodulin (CaM)-binding motif (35). The binding constants (Kd) for CaM differ between the isoforms (35). The constitutive NOS isoforms (eNOS and nNOS) transiently associate with CaM. In contrast, the inducible isoform (iNOS) forms a very tight complex with CaM (35). Correspondingly, eNOS and nNOS activities are tightly controlled by cytosolic Ca(II) fluxes while iNOS is constitutively active (35).
1.3 The Mammalian NO/cGMP Signal Transduction Pathway: NO Detection and Soluble Guanylate Cyclase
1.3.1 Soluble guanylate cyclase
In the NO/cGMP cell signaling pathway, the receptor for NO is soluble guanylate cyclase (sGC) (36-38). This enzyme belongs to the larger class of nucleotide cyclase enzymes including: adenylate cyclase (AC), particulate guanylate cyclase (pGC), and sGC (39). sGC participates in the cGMP dependent NO signaling pathway as follows: a generator cell utilizes the enzyme NOS to produce a concentration of NO, which then diffuses from its site of generation, through the tissue, until it reaches a target cell. Upon reaching a target cell, NO binds to its cognate receptor, sGC (Figure 1.5). As an NO dependent response, sGC catalyzes the cyclization of guanosine 5’-triphosphate (GTP) to give 3’, 5’-cyclic guanosine monophosphate (cGMP) and pyrophosphate (PPi) (Figure 1.5) (40). The cGMP produced by this reaction elicits cellular effects through three pathways: cGMP positively regulates the activity cGMP-dependent protein kinases such as protein kinase G (PKG) (41); cGMP activates a class of cyclic nucleotide phosphodiesterases (42, 43); and cGMP regulates the conductance of cGMP-gated ion channels such as the cyclic nucleotide-gated channels (CNG) of the vertebrate phototransduction system (44). Hence, sGC is a central hub in cell signaling pathways, transducing intercellular signals carried by NO into intracellular responses.
1.3.2 Domain architecture
sGCs are dimeric proteins, in which the two polypeptide subunits may be identical or not and have associated cofactors. While the major function of sGC in mammals is as a signal transducer in the NO/cGMP signal transduction pathway, related soluble guanylate cyclases also exist in non-mammalian organisms (e.g. Drosophila melanogaster (Dm) and Caenorhabditis elegans (Ce)) (45, 46). In these, sGC may respond to other diatomic gases such as molecular oxygen (O2) (46). Catalytically competent sGC enzymes may exist either as heterodimers such as the a1/b1 isoform purified from bovine lung tissues (38) or as homodimers such as Gyc-88E from Dm (47, 48). Each sGC subunit displays a similar domain architecture comprised of four functional units (Figure 1.6) (40): a conserved N-terminal regulatory domain, the heme-oxygen nitric oxide binding (H-NOX) motif in both subunits of homodimeric sGCs and in the b1 subunit of heterodimeric sGCs (5); a central element consisting of a per-ARNT-Sim (PAS) domain; an amphipathic helical coiled-coil segment (2); and a C-terminal nucleotide cyclase domain (49). No high resolution X-ray crystal structures of full length sGC have been solved. Thus, insights into the structure and function of this enzyme draw upon structures derived for related proteins and protein domains as well as biochemical experiments.
1.3.3 Guanylate cyclase domain
The cyclization reaction of GTP to give cGMP and PPi is catalyzed by the C-terminal guanylate cyclase domain of sGC (49). Based upon sequence phylogeny within the catalytic core, members of the purine nucleotidyl cyclase protein family are grouped into separate classes, I-VI (50). Together with adenylate cyclases (AC), eukaryotic guanylate cyclase (GC) domains belong to the third class of nucleotide cyclases (39). High-resolution structural studies by X-ray crystallography have provided atomic resolution snapshots of two representative members of the (class III) nucleotide cyclase family (Figure 1.7): AC (7, 51, 52) and GC (6, 53). These structures show that the class III nucleotide cyclases share a common dimeric organization. The two subunits associate in a “head-to-tail” manner, with the active site is situated at the interface of these two subdomains and residues from both monomers contributing to it. The detailed architecture of the catalytic sites differs between homodimeric and heterodimeric class III nucleotide cyclases. Homodimeric GCs, such as Cya2, possess two symmetric active sites (6), while heterodimeric ACs and GCs such as rat AC and the a1/b1 isoform of sGC contain two pseudosymmetric sites with only one of the two sites being catalytically active (49). In heterodimeric cyclases, the second pseudosymmetric site may bind small molecules that modulate the enzyme’s activity. For example, the natural product forskolin, a pharmacological modulator of adenylate cyclase, binds to this enzyme in its pseudosymmetric site (7).
1.3.4 Central domains
The central region of sGC (residues 259-364 in the a subunit and residues 204-408 in the b subunit) is a member of the per-ARNT-Sim (PAS) protein domain family (54, 55). Sequence profile searches and phylogenetic analyses by Iyer and colleagues showed that this domain is frequently found in association with the H-NOX structural motif either in the context of multi-domain signal transduction proteins in eukaryotes or in the context of multi-gene signal transduction operons in prokaryotes (56). Because of this genomic association with the H-NOX domain, this motif was given the name heme nitric oxide binding associated (HNOBA) domain (56). Two lines of evidence support this classification of the central domain of sGC into the PAS family. First, secondary structure prediction algorithms and sequence-structure threading methods suggest that this domain possesses the topology previously observed in X-ray crystal structures of representative members of the PAS family (eg. EcDOS, AvNifL, and RmFixL), a 7-stranded anti-parallel b-barrel flanked by several a-helices (Figure 1.8) (56-58). Second, a recent X-ray crystal structure of a prokaryotic HNOBA domain illustrated that one particular prokaryotic HNOBA domain adopts a PAS fold (2). A sequence of 50-60 amino acids located C-terminal to the PAS/HNOBA domain and N-terminal to the cyclase domain forms a coiled-coil (59, 60). In concert with the coiled-coil domain and structural elements of the cyclase domain, the HNOBA domain of sGC mediates sGC subunit dimerization (2, 61, 62).
1.3.5 H-NOX domain
The sGC H-NOX domain, consisting of the first ~200 amino acids of the b subunit of heterodimeric and of the single subunit of homodimeric sGCs, binds a protoporphyrin IX heme cofactor (Figure 1.9) (46, 56, 61, 63, 64). The bound heme cofactor endows the enzyme with the ability to bind and sense diatomic gases dissolved in solution. For example in vitro, the cGMP synthase activity of the a1/b1 isoform of rat sGC is enhanced ~100-200 fold by the binding of NO (38). As isolated, the H-NOX domain contains a ferrous (Fe(II)), pentacoordinate, high-spin (electronic spin, S = 2) heme (38). Several independent lines of evidence support this assignment. The electronic absorption spectrum of sGC has a Soret band at 431 nm and a single a/b band, similar to that of hemoglobin (38, 63). By electron paramagnetic resonance (EPR), the enzyme is silent (65). In the absence of an external reductant, sGC readily forms a complex with CO (Fe(II)-CO) with a Soret band at 423 nm (66). Resonance Raman spectra, which can report on heme oxidation state, coordination, and spin state, also support the Fe(II), pentacoordinate assignment (67). Mutagenesis studies of the rat b1 isoform have shown that the sole axial ligand of the sGC heme cofactor is a histidine residue (residue H105) (68, 69). Thus, the heme cofactor of sGC resembles myoglobin with respect to its oxidation and ligation state.
1.3.6 Ligand chemistry of the H-NOX domain
Binding of NO to the heme of sGC increases the enzyme’s activity; following addition of NO to sGC, the cGMP synthase activity increases two orders of magnitude above the basal activity level (38). Early biochemical evidence supported the hypothesis that NO activates sGC by forming a nitrosyl-heme (Fe(II)-NO) complex. Activation with NO is dependent upon heme (37, 70). Heme-deficient enzyme is not activated to the same extent as enzyme containing heme. Furthermore, addition of preformed Fe(II)-NO complexes to the heme-deficient protein activates its enzymatic activity (37, 71). However, the activation mechanism of sGC does not solely rely on binding of NO to the heme. Recent evidence indicates that while the binding of NO to the heme of sGC is necessary for enzyme activation, it is not sufficient (72). Binding of NO to a second site on the enzyme of an unknown chemical nature may be required for full activity (73). As evidenced by electronic absorption spectra (38) and EPR spectra (65), NO binding leads to the cleavage of the heme iron-histidine bond, resulting in the formation of a 5-coordinate Fe(II)-NO complex. 5-coordinate Fe(II)-NO complexes typically have Soret bands at 399 nm and split a/b bands (38). In contrast, hexacoodinate Fe(II)-NO complexes typically have Soret bands at 420 nm (11). The structural changes associated with the binding of NO to the H-NOX domain of sGC and the resultant cleavage reaction of the axial histidine iron bond are at present unknown. However, a structure-activity relationship study of Ignarro and coworkers provided evidence that the cleavage of the axial histidine iron bond is an important component of the activation mechanism of the enzyme (74). In that study, the authors showed that protoporphyrin IX without any bound metal activates sGC in a similar manner as Fe(II)-NO complexes, thereby suggesting that the cleavage of the iron-histidine bond is an important component of sGC activation.
The heme cofactor of soluble guanylate cyclase also readily binds carbon monoxide (CO). Upon binding CO via the sGC heme cofactor, the activity of sGC is enhanced 4 fold over basal activity (38). While the physiological relevance of CO signal transduction in vivo is still a matter of open debate (75), CO is an important chemical tool for studying the biophysical properties of hemoproteins. For example in the context of vibrational spectroscopies, (e.g. infrared and resonance Raman), characteristic bond stretching frequencies of heme-CO complexes in proteins report on the structural properties of the surrounding protein environment (76-78). By electronic absorption spectroscopy, the Fe(II)-CO sGC complex has a Soret peak at 423 nm and a split a/b signal (38). This spectrum indicates a 6-coordinate complex with CO as the first axial ligand and imidazole as the second (66). Based upon resonance Raman spectroscopy, the heme cofactor is predominantly in a 6-coordinate configuration (67). Thus, together these data suggest that Fe(II)-CO sGC possesses an axial histidine iron ligand similar to that observed in the apo- Fe(II) state.
1.4 H-NOX domains
1.4.1 Heme based sensors
Proteins that detect diatomic gases play vital roles in mediating events in the cell. For example, metazoan sGCs play a role in nitric oxide (NO) and oxygen (O2)dependent signal transduction pathways by coordinating the respective gases (46, 79, 80). Diatomic gas sensor proteins typically contain two distinct domains: a heme-containing sensor domain that binds diatomic ligands and an effector domain that generates an output signal (81). Common to these proteins are heme iron cofactors whose coordination properties are precisely tuned by the active site of the protein to bind a ligand with high specificity (81-83). For example, sGC binds NO at picomolar concentrations even in the presence of micromolar concentrations of oxygen (38, 84). It is this unique property of sGC that enables the enzyme to act as a specific NO sensor. Though diatomic gas binding proteins are integral components of biological signaling systems, their functions are not completely understood at the molecular level. In particular, structural knowledge of the protein environment surrounding the heme, while essential to an understanding of the mechanisms by which gas sensors such as sGC discriminate between ligands, is incomplete.
Recently, it was recognized that the N-terminal heme domain of sGC belongs to a larger family of proteins, the H-NOX domains (Heme Nitric Oxide/OXygen) (56). H-NOX proteins share spectroscopic properties, sequence homology (15-40 % identity), and key conserved residues with sGC. These observations suggest that members of this family provide excellent structural and biochemical models of the heme domain of sGC (85, 86). Although H-NOX domains are present in the genomes of both prokaryotes and eukaryotes, the functions of these signaling modules in the cell are likely diverse given their distinct biochemical properties (87). For instance, with respect to their heme chemistry, the various members of this protein family have different ligand binding properties (84, 87). Specifically, SO2144 from Shewanella oneidensis (So) and VCA0720 from Vibrio cholerae (Vc) exhibit a nearly identical discrimination for NO over O2 and may therefore act as NO sensors under physiological conditions (85, 88). In contrast, Tar4H from Thermoanaerobacter tengcongensis (Tt) and GCY-35 from Ce both bind O2 and NO and have ligand-binding properties similar to those of hemoglobin (46, 85). In vivo, GCY-35 regulates aerotaxis in Ce (46). These observations support the hypothesis that some H-NOX proteins act as O2 sensors. In prokaryotes, oxygen binding and non-oxygen binding H-NOX domains are found in different genomic contexts (Figure 1.10). In the original bioinformatics study of Iyer et al., a series of PSI-BLAST searches were performed using the heme domain of sGC (b1 1-194) (56). Eleven predicted open reading frames (ORFs) were found in facultative aerobic bacteria, typically found in association with histidine kinase proteins or histidine kinase/response regulator protein pairs (56). Five predicted ORFs were identified in obligate anaerobes as N-terminal domains of larger, multi-domain, methyl-accepting chemotaxis proteins (MCPs) (56).
1.4.2 H-NOX domain structure
Crystal structures of representative members of the H-NOX family were solved recently, an oxygen binding H-NOX domain in 2004 (5, 89) and a non-oxygen binding H-NOX domain in 2007 (11). The first of these structures was of Tar4H, an H-NOX domain from Tt that forms six coordinate complexes with O2, CO, and NO (5, 85). The structure of this protein was solved at 1.77 Å resolution and revealed the molecular functions of many of the residues that are strictly conserved in the H-NOX family of proteins (Figure 1.11) (5, 85). First, a set of hydrogen bonds are present between residues Y132, S134, and R136, the “YxSxR motif,” and the proprionate side chains of the heme moiety (5). Site-directed mutagenesis/enzymatic activity relationships support the idea that these non-covalent protein-heme contacts are also present in mammalian sGCs (90, 91). Second, the imidazole side chain of H102 coordinates to one of the two axial heme iron positions; thus, this residue is the heme proximal ligand (5). Mutations of the corresponding residue in sGC (H105) result in the loss of the ability to bind the heme cofactor. Moreover, the sGC H105G mutant requires imidazole to properly fold and bind heme (69) and the sGC H105F mutant does not bind heme in any known conditions (68).
In the Tar4H structure (Figure 1.12A), a hydrogen bond is formed between the bound O2 and the Y140 sidechain on the opposite distal side of the heme as the axial residue H102 residue. The ligand-binding differences between the NO binding and O2 binding classes of H-NOX domains suggest that with a relatively small number of amino acid substitutions H-NOX proteins are able to discriminate between binding NO and O2 ligands (87, 92). Thus, comparative structural analyses of H-NOX proteins reveal how the protein tunes the coordination properties of heme iron. Indeed, while a hydrogen bonding triad of residues is present in the distal heme pocket of Tar4H (5) the distal heme pocket of the non-oxygen binding H-NOX domain from the cyanobacteria Nostoc sp PCC 7120 (Ns) lacks such a set of residues (Figure 1.12B) (11). Instead, the distal heme pocket of this protein is comprised of hydrophobic residues such as I, L, F, and V residues. Homology models of the heme pocket of sGC suggest the distal heme pocket of this enzyme shares a similar hydrophobic architecture (93). As Fe(II) heme-O2 complexes resemble Fe(III) heme-O2– species with respect to the distribution of negative charge, the presence of hydrogen bonding residues in the H-NOX distal pocket are likely required by the protein to stabilize the corresponding complex by charge neutralization (94). Thus, by primary sequence analysis and homology modeling, a heuristic algorithm for H-NOX ligand binding characteristics can be constructed. If hydrogen bonding residues are predicted to be present in the H-NOX distal pocket, the domain will likely stably bind oxygen. Conversely, if such residues are absent, the domain will not stably bind oxygen at its heme cofactor. This mechanistic proposal is supported by comparative ligand binding kinetic studies of H-NOX domains (92). For example, the I145Y mutant of the sGC heme domain construct b1 1-385 is able to bind oxygen while the wild type protein does not (92).
The heme cofactor of the Tar4H H-NOX domain is distorted from planarity (5). Based upon extensive studies of small heme model compounds, the tetrapyrrole ring of the heme group is aromatic and adopts a planar configuration in the absence of bulky substituents on the ring that cause steric crowding (95-100). Typically, nonplanar heme configurations are higher in energy than planar configurations (101). However, when placed within the matrix of a protein, heme cofactors may deviate significantly from planarity (102-105). Residues that distort the heme from planarity are often conserved between homologous proteins from different species, suggesting that heme distortion plays important biochemical functions (106). Accordingly, mutation of a strictly conserved proline residue (P115) to alanine (P115A) in the heme binding pocket of Tar4H H-NOX flattens the heme cofactor (107). Decreasing heme distortion in Tar4H increases affinity for oxygen and decreases the reduction potential of the heme iron (107). Additionally, flattening of the heme is associated with significant shifts in the position of the N-terminus of the protein (107). These observations show a clear link between the heme conformation and Tar4H H-NOX structure. Also, heme distortion is an important property of the Tar4H H-NOX domain that modulates the molecule’s biophysical properties. As P115 is strictly conserved, it may also be involved in heme distortion in other members of the H-NOX family (5). Thus, heme distortion may be a general feature of the H-NOX family of proteins, participating in their chemical mechanism of cell signaling.
1.5 Candidate H-NOX Domains for Further Structural Study
The crystal structure of Tar4H, a protein from Tt, provided important insights into the overall topology of the H-NOX domain family (5) and laid a foundation for structural models of the diatomic gas ligand binding module of mammalian sGCs. Furthermore, mutagenesis studies guided by the X-ray structure of this molecule established a molecular basis for ligand discrimination (92). However, these studies do not provide insights into the conformational transitions that H-NOX proteins such as sGC make during the course of cell signaling because multiple structures in different states of signal transduction activity are not available for any H-NOX domain based signaling system characterized to date. Thus, the following question remains unanswered: how do H-NOX proteins bind diatomic gases at their heme iron cofactors and transduce free energy associated with ligand binding into cell signaling pathways? To address this question, multiple high-resolution structures of an H-NOX protein in different states of signaling activity are required. In this thesis, solution nuclear magnetic resonance (NMR) spectroscopy studies of the H-NOX protein SO2144 from So are reported in an effort to address this question.
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