Is histidine dissociation a critical component of the NO/H-NOX signaling mechanism? Insights from X-ray absorption spectroscopy

The H-NOX (Heme-Nitric oxide/OXygen binding) family of diatomic gas sensing hemoproteins has attracted great interest. Soluble guanylate cyclase (sGC), the well-characterized eukaryotic nitric oxide (NO) sensor is an H-NOX family member. When NO binds sGC at the ferrous histidine-ligated protoporphyrin-IX, the proximal histidine ligand dissociates, resulting in a 5-coordinate (5c) complex; formation of this 5c complex is viewed as necessary for activation of sGC. Characterization of other H-NOX family members has revealed that while most also bind NO in a 5c complex, some bind NO in a 6-coordinate (6c) complex or as a 5c/6c mixture. To gain insight into the heme pocket structural differences between 5c and 6c Fe(ii)-NO H-NOX complexes, we investigated the extended X-ray absorption fine structure (EXAFS) of the Fe(II)-unligated and Fe(II)-NO complexes of H-NOX domains from three species, Thermoanaerobacter tengcongensis, Shewanella woodyi, and Pseudoalteromonas atlantica. Although the Fe(II)-NO complex of TtH-NOX is formally 6c, we found the Fe-N(His) bond is substantially lengthened. Furthermore, although NO binds to SwH-NOX and PaH-NOX as a 5c complex, consistent with histidine dissociation, the EXAFS data do not exclude a very weakly associated histidine. Regardless of coordination number, upon NO-binding, the Fe-N(porphyrin) bond lengths in all three H-NOXs contract by ~0.07 ?. This study reveals that the overall heme structure of 5c and 6c Fe(II)-NO H-NOX complexes are substantially similar, suggesting that formal histidine dissociation may not be required to trigger NO/H-NOX signal transduction. The study has refined our understanding of the molecular mechanisms underlying NO/H-NOX signaling.     

Experimental Determination of an Isolated trans-Dinitrosyl Manganese(II) Heme Analogue

The lack of direct proof in either natural or synthetic systems for trans-dinitrosyl hemes, a key intermediate in the reactions of heme proteins (e.g. soluble guanylate cyclase (sGC), cytochrome c′ and So H-NOX) with nitric oxide (NO), has hampered understanding of the exact reaction mechanisms, such as the formation of the five-coordinate heme complex with NO at the proximal side (5c NO_P). Herein, we report the first isolation of a dinitrosyl metalloporphyrin complex, the six-coordinate, low-spin {Mn(NO)₂}⁷ species [Mn(TPP)(NO)₂] (TPP²⁻=meso-tetraphenylporphyrin dianion). The complex shows distinct features, such as an elongated axial bond (1.877(9) vs. 1.641(5) Å), a higher NO stretching bond position (1760 vs. 1735 cm⁻¹) and an isotropic resonance at g = 2.0, in sharp contrast to those of five-coordinate mononitrosyl analogues. In situ diffuse reflectance infrared Fourier transform spectroscopy (DRIFT) and EPR studies provided deep insight into the reaction processes, demonstrating different responses of porphyrinates to NO.     

Binding of YC-1 or BAY 41-2272 to soluble guanylyl cyclase induces a geminate phase in CO photolysis

Soluble guanylyl/guanylate cyclase (sGC), a heme-containing heterodimeric protein of approximately 150 kDa, is the primary receptor for nitric oxide, an endogenous molecule of immense physiological importance to animals. Recent studies have identified compounds such as YC-1 and BAY 41-2272 that stimulate sGC independently of NO binding, properties of importance for the treatment of endothelial dysfunction and other diseases linked to malfunctioning NO signaling pathways. We have developed a novel expression system for sGC from Manduca sexta (the tobacco hornworm) that retains the N-terminal two-thirds of both subunits, including heme, but is missing the catalytic domain. Here, we show that binding of compounds YC-1 or BAY 41-2272 to the truncated protein leads to a change in the heme pocket such that photolyzed CO cannot readily escape from the protein matrix. Geminate recombination of the trapped CO molecules with heme takes place with a measured rate of 6 x 10(7) s(-1). These findings provide strong support for an allosteric regulatory model in which YC-1 and related compounds can alter the sGC heme pocket conformation to retain diatomic ligands and thus activate the enzyme alone or in synergy with either NO or CO.     

The Chemistry and Biology of Soluble Guanylate Cyclase Stimulators and Activators

The vasodilatory properties of nitric oxide (NO) have been utilized in pharmacotherapy for more than 130 years. Still today, NO‐donor drugs are important in the management of cardiovascular diseases. However, inhaled NO or drugs releasing NO and organic nitrates are associated with noteworthy therapeutic shortcomings, including resistance to NO in some disease states, the development of tolerance during long‐term treatment, and nonspecific effects, such as post‐translational modification of proteins. The beneficial actions of NO are mediated by stimulation of soluble guanylate cyclase (sGC), a heme‐containing enzyme which produces the intracellular signaling molecule cyclic guanosine monophosphate (cGMP). Recently, two classes of compounds have been discovered that amplify the function of sGC in a NO‐independent manner, the so‐called sGC stimulators and sGC activators. The most advanced drug, the sGC stimulator riociguat, has successfully undergone Phase III clinical trials for different forms of pulmonary hypertension.     

Molecular Dynamics Simulations of Metal Ion Binding to the His‐tag Motif

In our previous study, we have observed that the chelation of various metal ions to the His‐tag motifs mostly involves the i and i+2 His residues for Ni²⁺, Cu²⁺, Zn²⁺ and Co²+. In the present study, various 200 ps molecular dynamics simulations were further conducted to investigate the chelating pathway of various metal ions to the His‐tag motif with 6 His residues (His‐tag6) and the binding affinities of these metal binding pockets towards these metal ions. The results indicate that His‐tag6 with the chelated metal ion located in positions His(2,4) or His(3,5) exhibits the strongest affinity for Ni²+ and Cu²+.K⁺ was found to be preferred to chelate in His(1,3) and His(3,5) coordinations. However, Fe³+ was found to have higher affinity towards His(1,3) and His(2,4) binding pockets. Our results also suggest that Ni²⁺ exhibits the highest binding affinity towards His‐tag6 over the other metal ions. Most of the structural variations of the His‐tag6 motif were from the Histidyl side chains during metal ion binding. In addition, there is an inverse linear correlation between the final chelated distance and the charge/volume ratio of metal ion. There is a negative correlation between the metal binding affinity and the averaged potential energy generated from the MD simulations.     

Heme flattening is sufficient for signal transduction in the H-NOX family

The H-NOX family of nitric oxide (NO) sensing proteins has received considerable attention because its members include the mammalian NO sensor, soluble guanylate cyclase. Despite this attention, the mechanism of signal transduction has not been elucidated. Structural studies of bacterial members of the family have revealed that the H-NOX heme cofactor is extremely distorted from planarity. Furthermore, it has been determined that heme distortion is maintained primarily by a conserved proline residue located in the proximal heme pocket. It has been suggested that changes in heme planarity may contribute to signal transduction. Here we demonstrate that heme flattening is, indeed, sufficient for signal transduction in the H-NOX family. Using our previously described H-NOX/diguanylate cyclase functional partners from Shewanella woodyi, we demonstrate that mutation of the conserved proline (P117 in SwH-NOX) to alanine, which results in heme flattening, has the same affect on phosphodiesterase activity as NO binding to wildtype SwH-NOX. This study demonstrates, for the first time, that heme flattening mimics the activated, NO-bound state of H-NOX and suggests that NO binding induces heme flattening as part of the signal transduction mechanism in the H-NOX family.     

可溶性鸟苷酸环化酶介导NO信号转导的结构基础及其分子机制研究

可溶性鸟苷酸环化酶(sGC)是NO信号转导通路中的核心金属酶,是NO的敏感器和受体.sGC含有?和?两个亚基,每个亚基分别具有3个结构域,包括血红素结构域、中心结构域和催化结构域,两个亚基的血红素结构域共享有一个血红素,NO结合到sGC的血红素后,激活sGC,催化其底物GTP转化为二级信号分子cGMP,开启PKG信号通路,导致血管舒张.NO信号转导通路异常将导致多种疾病的发生,如多种心血管疾病、肺动脉高血压、心力衰竭及神经退行性疾病等.近20年来,关于sGC的结构、功能、激活机制及其在生理与病理中的作用有了很多进展.本文重点对sGC的结构、功能及其活化/失活机制研究进展进行综述.     

Nitric oxide interaction with cytochrome c' and its relevance to guanylate cyclase. Why does the iron histidine bond break?

Soluble guanylate cyclase (sGC), the mammalian receptor for nitric oxide (NO), is a heme protein with a histidine as the proximal ligand. Formation of a five-coordinate heme-NO complex with the associated Fe-His bond cleavage is believed to trigger a conformational change that activates the enzyme and transduces the NO signal. Cytochrome c' (cyt c') is a protobacteria heme protein that has several similarities with sGC, including the ability to form a five-coordinate NO adduct and the fact that it does not bind oxygen. Recent crystallographic characterization of cyt c' from Alcaligenes xylosoxidans (AXCP) has yielded the discovery that exogenous ligands are able to bind to the Fe center from either side of the porphyrin plane. In this paper, we explore the molecular basis of the NO interaction with AXCP using hybrid quantum-classical simulation techniques. Our results suggest that Fe-His bond breaking depends not only on the iron-histidine bond strength but also on the existence of a local minimum conformation of the protein with the histidine away from the iron. We also show that AXCP is a useful paradigm for NO interaction with heme proteins, particularly regarding the activation/deactivation mechanism of sGC. The results presented here fully support a recently proposed model of sGC activation in which NO is not only the iron ligand but also catalyzes the activation step.     

The roles of cysteines in the heme domain of human soluble guanylate cyclase

Soluble guanylate cyclase(sGC) is a critical heme-containing enzyme involved in NO signaling.The dimerization of sGC subunits is necessary for its bioactivity and its mechanism is a striking and an indistinct issue.The roles of heme domain cysteines of the sGC on the dimerization and heme binding were investigated herein.The site-directed mutations of three conserved cysteines(C78A,C122A and C174S) were studied systematically and the three mutants were characterized by gel filtration analysis,UV-vis spectroscopy and heme transfer examination.Cys78 was involved in heme binding but not referred to the dimerization,while Cys174 was demonstrated to be involved in the homodimerization.These results provide new insights into the cysteine-related dimerization regulation of sGC.     

Sensitive and selective detection of nitric oxide using an H-NOX domain

Tt Y140F, a mutant of the H-NOX (Heme-Nitric Oxide or OXygen binding domain) protein from Thermoanaerobacter tengcongensis, is presented as a novel genetically encoded NO sensor. Tt Y140F is easily purified and obtained in high yields from standard E. coli expression systems. It is extremely stable as both the ferrous unligated and ferrous-nitrosyl complexes in air over a range of temperatures (up to 70 degrees C) without oxidizing, denaturing, or binding O2. NO binding is quantitative and can be followed using simple absorbance spectroscopy. In theory, NO concentrations between 300 nM and 30 muM can be accurately and easily detected.     

Multiple Proton Confinement in the M2 Channel from the Influenza A Virus

The tetrameric M2 protein bundle of the influenza A virus is the proton channel responsible for the acidification of the viral interior, a key step in the infection cycle. Selective proton transport is achieved by successive protonation of the conserved histidine amino acids at position 37. A recent X-ray structure of the tetrameric transmembrane (TM) domain of the protein (residues 22-46) resolved several water clusters in the channel lumen, which suggest possible proton pathways to the His37 residues. To explore this hypothesis, we have carried out molecular dynamics (MD) simulations of a proton traveling towards the His37 side chains using MD with classical and quantum force fields. Diffusion through the first half of the channel to the "entry" water cluster near His37 may be hampered by significant kinetic barriers due to electrostatic repulsion. However, once in the entry cluster, a proton can move to one of the acceptor His37 in a nearly barrierless fashion, as evidenced both by MD simulations and a scan of the potential energy surface (PES). Water molecules of the entry cluster, although confined in the M2 pore and restricted in their motions, can conduct protons with a rate very similar to that of bulk water.     

Dynamic mechanism for the autophosphorylation of CheA histidine kinase: molecular dynamics simulations

The two-component system (TCS) is an important signal transduction component for most bacteria. This signaling pathway is mediated by histidine kinases via autophosphorylation between P1 and P4 domains. Taking chemotaxis protein CheA as a model of TCS, the autophosphorylation mechanism of the TCS histidine kinases has been investigated in this study by using a computational approach integrated homology modeling, ligand-protein docking, protein-protein docking, and molecular dynamics (MD) simulations. Four nanosecond-scale MD simulations were performed on the free P4 domain, P4-ATP, P4-TNPATP, and P1-P4-ATP complexes, respectively. Upon its binding to the binding pocket of P4 with a folded conformation, ATP gradually extends to an open state with help from a water molecule. Meanwhile, ATP forms two hydrogen bonds with His413 and Lys494 at this state. Because of the lower energy of the folded conformations, ATP shrinks back to its folded conformations, leading to the rupture of the hydrogen bond between ATP and Lys494. Consequently, Lys494 moves away from the pocket entrance, resulting in an open of the ATP lid of P4. It is the open state of P4 that can bind tightly to P1, where the His45 of P1 occupies a favorable position for its autophosphorylation from ATP. This indicates that ATP is not only a phosphoryl group donor but also an activator for CheA phosphorylation. Accordingly, a mechanism of the autophosphorylation of CheA is proposed as that the ATP conformational switch triggers the opening of the ATP lid of P4, leading to P1 binding tightly, and subsequently autophosphorylation from ATP to P1.     

A molecular dynamics investigation of the resting, hydrogen peroxide-bound and compound II forms of cytochrome C peroxidase and Artromyces ramosus peroxidase

Molecular Dynamics (MD) simulations have been used to study structural and dynamic properties of the resting, hydrogen peroxide adduct and compound II forms of cytochrome C peroxidase (CCP) and Artromyces ramosus peroxidase (ARP). MD simulations of CCP show that: (i) hydrogen peroxide might form an outer sphere complex within the active site of the enzyme before the coordination to the iron centre takes place; (ii) Trp51 and His52 residues play a crucial role in the recognition and binding of hydrogen peroxide, while Arg48 is not directly involved; (iii) distal histidine (His52) allows an easy proton 1,2 shift within the H₂O₂ molecule, while Arg48 is not expected to play a role as crucial as His52 in promoting the heterolytic O–O bond breaking; (iv) the large mobility (about 2 Å) of the side chain of Arg48 in the compound II form allows the formation of a hydrogen bond (H-bond) with the ferryl oxygen, which contributes to the stabilisation of such an intermediate. The active site of the ARP enzyme is characterised by structural and dynamic features slightly different from the CCP active site. In particular, (i) the outer sphere complex with hydrogen peroxide occurring in CCP is not observed in ARP because of the substitution of Trp51 of CCP with the more hydrophobic residue Phe55 of ARP; (ii) His56 and the carbonyl group of Arg52 are determinant in controlling the hydrogen peroxide binding and its orientation in the active site. In ARP, both H₂O₂ and His56 have orientation different than in CCP, but still suited for an easy 1,2 proton shift. (iii) Arg52 in ARP is on average more distant from the heme-iron than in CCP, but its relative orientation is suited to promote an easy cleavage of H₂O₂. (iv) In compound II form of ARP, the Arg52 side chain is too far from the oxy-ferryl group to form a hydrogen bond and therefore ARP looses a stabilising factor, which is present in the corresponding form of CCP.     

Theoretical density functional theory studies on interactions of small biologically active molecules with isolated heme group

We present ab-initio density functional theory studies on the interactions of small biologically active molecules, namely NO, CO, O(2), H(2)O, and NO(2) (-) with the full-size heme group. Our results show that the small molecule-iron bond is the strongest in carbonyl and the weakest in nitrite system. Trans influence induced by NO binding to the five-coordinate heme complex is shown. Nitric oxide in the resulting complex might be described as NO(-). The differences among the small ligands of XO type (CO, NO, O(2)), and their distant chemical behavior from H(2)O and NO(2) (-) ligands in binding to the Fe(II) ion, are shown. Moreover, the role of the heme ring as a reservoir of electrons in the studied complexes is invoked. The analysis of the parameters defining the iron-histidine bond indicates that this bond is longer and weaker in nitrosyl and carbonyl complexes than in the other systems. Our findings support the proposed mechanism of soluble guanylate cyclase (sGC) activation and suggest that the first step of sGC activation by CO may be the same as during the activation by NO. Obtained results are then compared with the data concerning smaller model of the heme, the porphyrin complexes, available in the literature.     

Prolonged exposure of chromaffin cells to nitric oxide down-regulates the activity of soluble guanylyl cyclase and corresponding mRNA and protein levels

Background

Soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide (NO) when the latter is produced at low concentrations. This enzyme exists mainly as a heterodimer consisting of one α and one β subunit and converts GTP to the second intracellular messenger cGMP. In turn, cGMP plays a key role in regulating several physiological processes in the nervous system. The aim of the present study was to explore the effects of a NO donor on sGC activity and its protein and subunit mRNA levels in a neural cell model.

Results

Continuous exposure of bovine adrenal chromaffin cells in culture to the nitric oxide donor, diethylenetriamine NONOate (DETA/NO), resulted in a lower capacity of the cells to synthesize cGMP in response to a subsequent NO stimulus. This effect was not prevented by an increase of intracellular reduced glutathione level. DETA/NO treatment decreased sGC subunit mRNA and β₁ subunit protein levels. Both sGC activity and β₁ subunit levels decreased more rapidly in chromaffin cells exposed to NO than in cells exposed to the protein synthesis inhibitor, cycloheximide, suggesting that NO decreases β₁ subunit stability. The presence of cGMP-dependent protein kinase (PKG) inhibitors effectively prevented the DETA/NO-induced down regulation of sGC subunit mRNA and partially inhibited the reduction in β₁ subunits.

Conclusions

These results suggest that activation of PKG mediates the drop in sGC subunit mRNA levels, and that NO down-regulates sGC activity by decreasing subunit mRNA levels through a cGMP-dependent mechanism, and by reducing β₁ subunit stability.     

Acetylcholinesterase: mechanisms of covalent inhibition of wild-type and H447I mutant determined by computational analyses

The reaction mechanisms of two inhibitors TFK+ and TFK0 binding to both the wild-type and H447I mutant mouse acetylcholinesterase (mAChE) have been investigated by using a combined ab initio quantum mechanical/molecular mechanical (QM/MM) approach and classical molecular dynamics (MD) simulations. In the wild-type mAChE, the binding reactions of TFK+ and TFK0 are both spontaneous processes, which proceed through the nucleophilic addition of the Ser203-Ogamma to the carbonyl-C of TFK+ or TFK0, accompanied with a simultaneous proton transfer from Ser203 to His447. No barrier is found along the reaction paths, consistent with the experimental reaction rates approaching the diffusion-controlled limit. By contrast, TFK+ binding to the H447I mutant may proceed with a different reaction mechanism. A water molecule takes over the role of His447 and participates in the bond breaking and forming as a "charge relayer". Unlike in the wild-type mAChE case, Glu334, a conserved residue from the catalytic triad, acts as a catalytic base in the reaction. The calculated energy barrier for this reaction is about 8 kcal/mol. These predictions await experimental verification. In the case of the neutral ligand TFK0, however, multiple MD simulations on the TFK0/H447I complex reveal that none of the water molecules can be retained in the active site as a "catalytic" water. Furthermore, our alchemical free energy calculation also suggests that the binding of TFK0 to H447I is much weaker than that of TFK+. Taken together, our computational studies confirm that TFK0 is almost inactive in the H447I mutant and also provide detailed mechanistic insights into the experimental observations.     

Molecular dynamics study of taxadiene synthase catalysis

Molecular dynamics (MD) simulations have been performed to study the dynamic behavior of noncovalent enzyme carbocation complexes involved in the cyclization of geranylgeranyl diphosphate to taxadiene catalyzed by taxadiene synthase (TXS). Taxadiene and the observed four side products originate from the deprotonation of carbocation intermediates. The MD simulations of the TXS carbocation complexes provide insights into potential deprotonation mechanisms of such carbocations. The MD results do not support a previous hypothesis that carbocation tumbling is a key factor in the deprotonation of the carbocations by pyrophosphate. Instead water bridges are identified which may allow the formation of side products via multiple proton transfer reactions. A novel reaction path for taxadiene formation is proposed on the basis of the simulations. © 2018 Wiley Periodicals, Inc.     

Theoretical study of the truncated hemoglobin HbN: exploring the molecular basis of the NO detoxification mechanism

Mycobacterium tuberculosis is the causative agent of human tuberculosis. The nitric oxide reaction with oxy-truncated hemoglobin N (trHbN) has been proposed to be responsible for the resistance mechanism by which this microorganism can evade the toxic effects of NO. In this work, we explore the molecular basis of the NO detoxification mechanism using a combination of classical and hybrid quantum-classical (QM-MM) simulation techniques. We have investigated the structural flexibility of the protein, the ligand affinity properties, and the nitric oxide reaction with coordinated O2. The analysis of the classical MD trajectory allowed us to identify Phe62 as the gate of the main channel for ligand diffusion to the active site. Moreover, the opening of the channel stems from the interplay between collective backbone motions and local rearrangements in the side chains of the residues that form the bottleneck of the tunnel. Even though the protein environment is not found to make a significant contribution to the heme moiety catalyzed reaction, the binding site influences the physiological function of the enzyme at three different levels. First, by isolating the intermediates formed in the reaction, it prevents nondesired reactions from proceeding. Second, it modulates the ligand (O2, NO) affinity of the protein, which can be ascribed to both distal and proximal effects. Finally, the stabilization of the Tyr33-Gln58 pair upon O2 binding might alter the essential dynamics of the protein, leading in turn to a mechanism for ligand-induced regulation.     

Molecular dynamics simulations of bovine cathepsin B and its complex with CA074

To promote our better understanding of the dynamic stability of the bovine cathepsin B structure, which is characterized by an extra disulfide bond at Cys148-Cys252 from the other species, and of the binding stability of CA074 (a cathepsin B-specific inhibitor), molecular dynamics (MD) simulations were performed for the enzyme and its CA074 complex, assuming a system in aqueous solution at 300 K. The MD simulation covering 400 ps indicated that the existence of a Cys148-Cys252 disulfide bond increases the conformational flexibility of the occluding loop, although the conformational stability of the overall structure is little affected. The structural characteristics of the complex elucidated by X-ray analysis were suggested to be also intrinsic and stable in the dynamic state; the hydrogen bonding/electrostatic interactions between the main and side chains of CA074 and the Sn and Sn' subsites of the enzyme were maintained throughout the MD simulation. Furthermore, the simulation made clear that the binding of CA074 significantly restricted the conformational flexibility of the substrate binding region, especially the occluding loop, of cathepsin B. Statistical analyses during the simulation suggest that the selectivity of CA074 for cathepsin B stems from the tight P1'-S1' and P2'-S2' interactions, assisted in particular by double hydrogen bonds between the carboxyl two oxygens of the CA074 C-terminus and the imidazole NH groups of His110 and His111 residues.     

Protein:Ligand binding free energies: A stringent test for computational protein design

A computational protein design method is extended to allow Monte Carlo simulations where two ligands are titrated into a protein binding pocket, yielding binding free energy differences. These provide a stringent test of the physical model, including the energy surface and sidechain rotamer definition. As a test, we consider tyrosyl‐tRNA synthetase (TyrRS), which has been extensively redesigned experimentally. We consider its specificity for its substrate l ‐tyrosine (l ‐Tyr), compared to the analogs d ‐Tyr, p‐acetyl‐, and p‐azido‐phenylalanine (ac‐Phe, az‐Phe). We simulate l ‐ and d ‐Tyr binding to TyrRS and six mutants, and compare the structures and binding free energies to a more rigorous “MD/GBSA” procedure: molecular dynamics with explicit solvent for structures and a Generalized Born + Surface Area model for binding free energies. Next, we consider l ‐Tyr, ac‐ and az‐Phe binding to six other TyrRS variants. The titration results are sensitive to the precise rotamer definition, which involves a short energy minimization for each sidechain pair to help relax bad contacts induced by the discrete rotamer set. However, when designed mutant structures are rescored with a standard GBSA energy model, results agree well with the more rigorous MD/GBSA. As a third test, we redesign three amino acid positions in the substrate coordination sphere, with either l ‐Tyr or d ‐Tyr as the ligand. For two, we obtain good agreement with experiment, recovering the wildtype residue when l ‐Tyr is the ligand and a d ‐Tyr specific mutant when d ‐Tyr is the ligand. For the third, we recover His with either ligand, instead of wildtype Gln. © 2015 Wiley Periodicals, Inc.