Understanding How H-NOX (Heme Nitric Oxide/Oxygen) domain works needs first clarifying how diatomic gases are relocated inside this sensing protein. A molecular-mechanics approach

H-NOX (Heme Nitric Oxide/Oxygen) domain has widespread occurrence, either standalone or associated with functional proteins, sending signals for functions that span from modulating vasodilation and neurotransmission with humans to competition and symbiosis with bacteria. Understanding how H-NOX works, and possibly intervening on degeneration for health purposes, needs first clarifying how diatomic gases are relocated through this protein in relation to the deeply buried heme. To this end, a biased form of molecular dynamics, i.e., Random Accelaration Molecular Dynamics (RAMD), is used by applying a randomly oriented tiny force to heme-dissociated CO of Nostoc sp. H-NOX, while changing randomly the direction of the force, if CO travels less than specified for the evaluated block. The result is that a large area of the protein, comprising amino acids from serine 44 to leucine 67 along two adjacent helices, offers a broad portal to CO from the surrounding medium to the deeply buried heme. Most traffic is concentrated through a channel lined by tyrosine 49, valine 52, and leucine 67. This modifies the picture drawn from mapping Xe cavities on pressurizing Nostoc sp. H-NOX with Xe gas. What is the main pathway with Xe-cavity mapping becomes a minor pathway with RAMD, and vice versa. The reason is that the fluctuating protein under MD creates clefts for CO slipping through, as it is expected to occur in nature.     

Theoretical investigation on the diatomic ligand migration process and ligand binding properties in non‐O2‐binding H‐NOX domain

The Nostoc sp (Ns) H‐NOX (heme‐nitric oxide or OXygen‐binding) domain shares 35% sequence identity with soluble guanylate cyclase (sGC) and exhibits similar ligand binding property with the sGC. Previously, our molecular dynamic (MD) simulation work identified that there exists a Y‐shaped tunnel system hosted in the Ns H‐NOX interior, which servers for ligand migration. The tunnels were then confirmed by Winter et al. [PNAS 2011;108(43):E 881–889] recently using x‐ray crystallography with xenon pressured conditions. In this work, to further investigate how the protein matrix of Ns H‐NOX modulates the ligand migration process and how the distal residue composition affects the ligand binding prosperities, the free energy profiles for nitric oxide (NO), carbon monooxide (CO), and O₂ migration are explored using the steered MDs simulation and the ligand binding energies are calculated using QM/MM schemes. The potential of mean force profiles suggest that the longer branch of the tunnel would be the most favorable route for NO migration and a second NO trapping site other than the distal heme pocket along this route in the Ns H‐NOX was identified. On the contrary, CO and O₂ would prefer to diffuse via the shorter branch of the tunnel. The QM/MM (quantum mechanics/molecular mechanics) calculations suggest that the hydrophobic distal pocket of Ns H‐NOX would provide an approximately vacuum environment and the ligand discrimination would be determined by the intrinsic binding properties of the diatomic gas ligand to the heme group. Proteins 2013; 81:1363–1376. © 2013 Wiley Periodicals, Inc.     

On Dioxygen and Substrate Access to Soluble Methane Monooxygenases: An all‐Atom Molecular Dynamics Investigation in Water Solution

In a preliminary exploration of the dummy model for diiron proteins, random‐acceleration molecular dynamics (RAMD) revealed that a pure four‐helix bundle structure, like hemerythrin, constitutes an efficient cage for dioxygen (O₂), which can only leave from defined, albeit very broad, gates. However, this well ordered structure does not constitute an archetype on which to compare O₂ permeation of other diiron proteins, like the complex of soluble methane monooxygenase hydroxylase with the regulatory protein (sMMOH‐MMOB). The reason is that with this complex, unlike hemerythrin, the four helices of the four‐helix bundle are heavily bent, and RAMD showed that most traps for O₂ lie outside them. It was also observed that, in spite of a nearly identical van der Waals radius for O₂ and the natural substrate CH₄, the latter behaves under RAMD as a bulkier molecule than O₂, requiring a higher external force to be brought out of sMMOH‐MMOB along trajectories of viable length. All that determined with sMMOH‐MMOB multiple gates and multiple pathways to each of them through several binding pockets, for both O₂ and CH₄. Of the two equally preferred pathways for O₂, at right angle with one another, one proved to be in accordance with the Xe‐atom mapping for sMMOH. In contrast, none of the pathways identified for CH₄ proved to be in accordance with such mapping, CH₄ looking for more open avenues instead.     

Structural insights into the molecular mechanism of H‐NOX activation

Nitric oxide (NO) signaling in mammals controls important processes such as smooth muscle relaxation and neurotransmission by the activation of soluble guanylate cyclase (sGC). NO binding to the heme domain of sGC leads to dissociation of the iron–histidine (Fe–His) bond, which is required for enzyme activity. The heme domain of sGC belongs to a larger class of proteins called H‐NOX (Heme‐Nitric oxide/OXygen) binding domains. Previous crystallographic studies on H‐NOX domains demonstrate a correlation between heme bending and protein conformation. It was unclear, however, whether these structural changes were important for signal transduction. Subsequent NMR solution structures of H‐NOX proteins show a conformational change upon disconnection of the heme and proximal helix, similar to those observed in the crystallographic studies. The atomic details of these conformational changes, however, are lacking in the NMR structures especially at the heme pocket. Here, a high‐resolution crystal structure of an H‐NOX mutant mimicking a broken Fe–His bond is reported. This mutant exhibits specific changes in heme conformation and major N‐terminal displacements relative to the wild‐type H‐NOX protein. Fe–His ligation is ubiquitous in all H‐NOX domains, and therefore, the heme and protein conformational changes observed in this study are likely to occur throughout the H‐NOX family when NO binding leads to rupture of the Fe–His bond.     

On the Pathways of Biologically Relevant Diatomic Gases through Proteins. Dioxygen and Heme Oxygenase from the Perspective of Molecular Dynamics

This work deals with dioxygen (O₂) binding sites and pathways through inducible human heme oxygenase (HO‐1). The experimentally known distal binding site 1, and sites 2–3 above it, could be reproduced by means of non‐deterministic random‐acceleration molecular‐dynamics (RAMD) simulations. In addition, RAMD revealed the proximal binding site 5, a deeply‐seated binding site 4, which lies behind heme, as well as a few gates communicating with the external medium. In getting from site 1 to the main gate, which lies on the protein front opposed to site 4, O₂ follows chiefly the shortest direct pathway. Less frequently, O₂ visits intermediate sites 2, 4, or 5 along longer pathways. A similarity between HO‐1, myoglobin, and cytoglobin in using, for diatomic gas delivery, the direct shortest pathway from the heme center to the surrounding medium, is emphasized. Otherwise, comparing other proteins and diatomic gases, each system reveals its peculiarities as to sites, gates, and pathways. Thus, relating these properties to the physiological functions of the proteins remains in general a challenge for future studies.     

On the Egress of Carbon Monoxide from Myoglobin

The pathways of escape of carbon monoxide (CO) from sperm whale myoglobin were investigated by means of a biased form of all‐atoms molecular dynamics (RAMD), whereby a weak, randomly oriented force is applied to the center of mass of CO. The force only persists if the direction taken by CO continues for a given period of time, otherwise a new direction is randomly chosen. A statistically significant number of RAMD runs gave distinct responses according to the level of approximations used for the model. Thus, with rigid bonds to all H‐atoms, several portals for CO egress toward the solvent, besides the main H64 gate, were identified, like in recently published unbiased massive MD, six orders of magnitude acceleration of CO escape in RAMD notwithstanding. In contrast, by removing the approximation of rigid bonds in the model, only one of these extra portals was identified, besides the H64 portal. Sticking to this all‐free‐bonds model, Perutz's early suggestion that the H64 imidazole must rotate ‘out’ toward the solvent in order that CO can cross the H64 gate was directly implemented. RAMD Simulations with this model led to CO egress from the H64 gate only, reconciling theory with experiments.     

Amyloid β‐induced astrogliosis is mediated by β1‐integrin via NADPH oxidase 2 in Alzheimer's disease

Astrogliosis is a hallmark of Alzheimer′s disease (AD) and may constitute a primary pathogenic component of that disorder. Elucidation of signaling cascades inducing astrogliosis should help characterizing the function of astrocytes and identifying novel molecular targets to modulate AD progression. Here, we describe a novel mechanism by which soluble amyloid‐β modulates β1‐integrin activity and triggers NADPH oxidase (NOX)‐dependent astrogliosis in vitro and in vivo. Amyloid‐β oligomers activate a PI3K/classical PKC/Rac1/NOX pathway which is initiated by β1‐integrin in cultured astrocytes. This mechanism promotes β1‐integrin maturation, upregulation of NOX2 and of the glial fibrillary acidic protein (GFAP) in astrocytes in vitro and in hippocampal astrocytes in vivo. Notably, immunochemical analysis of the hippocampi of a triple‐transgenic AD mouse model shows increased levels of GFAP, NOX2, and β1‐integrin in reactive astrocytes which correlates with the amyloid β‐oligomer load. Finally, analysis of these proteins in postmortem frontal cortex from different stages of AD (II to V/VI) and matched controls confirmed elevated expression of NOX2 and β1‐integrin in that cortical region and specifically in reactive astrocytes, which was most prominent at advanced AD stages. Importantly, protein levels of NOX2 and β1‐integrin were significantly associated with increased amyloid‐β load in human samples. These data strongly suggest that astrogliosis in AD is caused by direct interaction of amyloid β oligomers with β1‐integrin which in turn leads to enhancing β1‐integrin and NOX2 activity via NOX‐dependent mechanisms. These observations may be relevant to AD pathophysiology.     

Binding Pockets and Permeation Channels for Dioxygen through Cofactorless 3‐Hydroxy‐2‐methylquinolin‐4‐one 2,4‐Dioxygenase in Association with Its Natural Substrate, 3‐Hydroxy‐2‐methylquinolin‐4(1H)‐one. A Perspective from Molecular Dynamics Simulations

This work describes an investigation of pathways and binging pockets (BPs) for dioxygen (O₂) through the cofactorless oxygenase 3‐hydroxy‐2‐methylquinolin‐4‐one 2,4‐dioxygenase in complex with its natural substrate, 3‐hydroxy‐2‐methylquinolin‐4(1H)‐one, in aqueous solution. The investigation tool was random‐acceleration molecular dynamics (RAMD), whereby a tiny, randomly oriented external force is applied to O₂ in order to accelerate its movements. In doing that, care was taken that the external force only continues, if O₂ moves along a direction for a given period of time, otherwise the force changed direction randomly. Gates for expulsion of O₂ from the protein, which can also be taken as gates for O₂ uptake, were found throughout almost the whole external surface of the protein, alongside a variety of BPs for O₂. The most exploited gates and BPs were not found to correspond to the single gate and BP proposed previously from the examination of the static model from X‐ray diffraction analysis of this system. Therefore, experimental investigations of this system that go beyond the static model are urgently needed.     

On Dioxygen Permeation through a Dehydrogenase‐Pyrroloquinoline Quinone Complex. A Molecular‐Dynamics Investigation

In this work, an all atom model of the quinoprotein dehydrogenase PqqC in complex with the PQQ (=4,5‐dihydro‐4,5‐dioxo‐1H‐pyrrolo[2,3‐f]quinoline‐2,7,9‐tricarboxylic acid) cofactor and dioxygen (O₂), solvated with TIP3 water in periodic boxes, was subjected to random‐acceleration molecular dynamics (RAMD). It was found that O₂ leaves the active binding pocket, in front of PQQ, to get to the solvent, as easily as with a variety of other O₂‐activating enzymes, O₂ carriers, and gas‐sensing proteins. The shortest pathway, orthogonal to the center of the mean plane of PQQ, was largely preferred by O₂ over pathways slightly deviating from this line. These observations challenge the interpretation of an impermeable active binding pocket of PqqC‐PQQ, as drawn from both X‐ray diffraction data of the crystal at low temperature and physiological experimentation.     

Unveiling the Pathways of Dioxygen Through the C2 Component of the Environmentally Relevant Monooxygenase p‐Hydroxyphenylacetate Hydroxylase from Acinetobacter baumannii: A Molecular Dynamics Investigation

In this work, models of the homotetrameric C2 component of the monooxygenase p‐hydroxyphenylacetate hydroxylase from Acinetobacter baumannii, in complex with dioxygen (O₂) and, or not, the substrate p‐hydroxyphenylacetate (HPA) were built. Both models proved to be amenable to random‐acceleration molecular dynamics (RAMD) simulations, whereby a tiny randomly oriented external force, acting on O₂ at the active site in front of flavin mononucleotide (FMNH^?), accelerated displacement of O₂ toward the bulk solvent. This allowed us to carry out a sufficiently large number of RAMD simulations to be of statistical significance. The two systems behaved very similarly under RAMD, except for O₂ leaving the active site more easily in the absence of HPA, but then finding similar obstacles in getting to the gate as when the active site was sheltered by HPA. This challenges previous conclusions that HPA can only reach the active center after that the C4aOOH derivative of FMNH^? is formed, requiring uptake of O₂ at the active site before HPA. According to these RAMD simulations, O₂ could well get to FMNH^? also in the presence of the substrate at the active site.     

Gates and Binding Pockets for Nitric Oxide with Cytochrome c′, According to Molecular Dynamics

Random‐acceleration molecular‐dynamics (RAMD) simulations with models of homodimeric 6‐ligated distal‐NO and 5‐ligated proximal‐NO cytochrome c′ complexes, in TIP3 H₂O, showed two distinct, non‐intercommunicating worlds. In the framework of a long cavity formed by four protein helices with heme at one extremity, NO was observed to follow different pathways with the two complexes to reach the solvent. With the 6‐ligated complex, NO was observed to progress by exploiting protein internal channels created by thermal fluctuations, and be temporarily trapped into binding pockets before reaching the preferred gate at the heme end of the cavity. In contrast, with the 5‐ligated complex, NO was observed to surface the solvent‐exposed helix 7, up to a gate at the other extremity of the protein, only occasionally finding an earlier, direct way out toward the solvent. That only bulk NO gets involved in forming the 5‐ligated proximal‐NO complex is in agreement with previous experimental observations, while the occurrence of binding pockets suggests that also reservoir NO might play a role with the distal‐NO complex.     

Structural characterization of the heme‐based oxygen sensor,AfGcHK, its interactions with the cognate response regulator,and their combined mechanism of action in a bacterial two‐component signaling system

The oxygen sensor histidine kinase AfGcHK from the bacterium Anaeromyxobacter sp. Fw 109‐5 forms a two‐component signal transduction system together with its cognate response regulator (RR). The binding of oxygen to the heme iron of its N‐terminal sensor domain causes the C‐terminal kinase domain of AfGcHK to autophosphorylate at His183 and then transfer this phosphate to Asp52 or Asp169 of the RR protein. Analytical ultracentrifugation revealed that AfGcHK and the RR protein form a complex with 2:1 stoichiometry. Hydrogen‐deuterium exchange coupled to mass spectrometry (HDX‐MS) suggested that the most flexible part of the whole AfGcHK protein is a loop that connects the two domains and that the heme distal side of AfGcHK, which is responsible for oxygen binding, is the only flexible part of the sensor domain. HDX‐MS studies on the AfGcHK:RR complex also showed that the N‐side of the H9 helix in the dimerization domain of the AfGcHK kinase domain interacts with the helix H1 and the β‐strand B2 area of the RR protein's Rec1 domain, and that the C‐side of the H8 helix region in the dimerization domain of the AfGcHK protein interacts mostly with the helix H5 and β‐strand B6 area of the Rec1 domain. The Rec1 domain containing the phosphorylable Asp52 of the RR protein probably has a significantly higher affinity for AfGcHK than the Rec2 domain. We speculate that phosphorylation at Asp52 changes the overall structure of RR such that the Rec2 area containing the second phosphorylation site (Asp169) can also interact with AfGcHK. Proteins 2016; 84:1375–1389. © 2016 Wiley Periodicals, Inc.     

Crystal structure of the Alpha subunit PAS domain from soluble guanylyl cyclase

Soluble guanylate cyclase (sGC) is a heterodimeric heme protein of ～150 kDa and the primary nitric oxide receptor. Binding of NO stimulates cyclase activity, leading to regulation of cardiovascular physiology and providing attractive opportunities for drug discovery. How sGC is stimulated and where candidate drugs bind remains unknown. The α and β sGC chains are each composed of Heme‐Nitric Oxide Oxygen (H‐NOX), Per‐ARNT‐Sim (PAS), coiled‐coil and cyclase domains. Here, we present the crystal structure of the α₁ PAS domain to 1.8 Å resolution. The structure reveals the binding surfaces of importance to heterodimer function, particularly with respect to regulating NO binding to heme in the β₁ H‐NOX domain. It also reveals a small internal cavity that may serve to bind ligands or participate in signal transduction.     

On the Pathways for CO Egress from Carboxy Human Cytoglobin. A Molecular‐Dynamics Investigation

This work discloses two bona fide gates through which the CO ligand can leave the distal cavity of carboxy human cytoglobin, reaching the solvent. The investigation was based on molecular dynamics, aided by a minimal randomly‐oriented force applied to the ligand. The shortest pathway progresses toward the main gate, H81‐R84, in the open state, with the H81 imidazole moiety turned toward the solvent. A longer pathway develops toward the diametrically opposed W31‐W151 gate. In between, CO may be entrapped into binding cavities, either along the path toward the gates, or in a cul‐de‐sac, from which CO may even be incapable to escape. This behavior contrasts with carboxy myoglobin, where the corresponding H64 gate, when opened, is the sole used by CO to get to the solvent. These observations, which could hold also for other small ligands of biological interest, such as O₂, NO, and NO$\rm{{_{3}^{-}}}$ , provide an answer to a neglected aspect of the mysterious six‐coordinated globins.     

H‐NOX regulation of c‐di‐GMP metabolism and biofilm formation in Legionella pneumophila

Haem Nitric oxide/OXygen (H‐NOX) binding domains are a family of haemoprotein sensors that are widespread in bacterial genomes, but limited information is available on their function. Legionella pneumophila is the only prokaryote found, thus far, to encode two H‐NOX proteins. This paper presents data supporting a role for one of the L. pneumophila H‐NOXs in the regulation of biofilm formation. In summary: (i) unmarked deletions in the hnox1 gene do not affect growth rate in liquid culture or replication in permissive macrophages; (ii) the Δhnox1 strain displays a hyper‐biofilm phenotype; (iii) the gene adjacent to hnox1 is a GGDEF‐EAL protein, lpg1057, and overexpression in L. pneumophila of this protein, or the well‐studied diguanylate cyclase, vca0956, results in a hyper‐biofilm phenotype; (iv) the Lpg1057 protein displays diguanylate cyclase activity in vitro and this activity is inhibited by the Hnox1 protein in the Fe(II)‐NO ligation state, but not the Fe(II) unligated state; and (v) consistent with the Hnox1 regulation of Lpg1057, unmarked deletions of lpg1057 in the Δhnox1 background results in reversion of the hyper‐biofilm phenotype back to wild‐type biofilm levels. Taken together, these results suggest a role for hnox1 in regulating c‐di‐GMP production by lpg1057 and biofilm formation in response to NO.     

Crystal structures of halohydrin hydrogen‐halide‐lyases from Corynebacterium sp. N‐1074

Halohydrin hydrogen‐halide‐lyase (H‐Lyase) is a bacterial enzyme that is involved in the degradation of halohydrins. This enzyme catalyzes the intramolecular nucleophilic displacement of a halogen by a vicinal hydroxyl group in halohydrins to produce the corresponding epoxides. The epoxide products are subsequently hydrolyzed by an epoxide hydrolase, yielding the corresponding 1, 2‐diol. Until now, six different H‐Lyases have been studied. These H‐Lyases are grouped into three subtypes (A, B, and C) based on amino acid sequence similarities and exhibit different enantioselectivity. Corynebacterium sp. strain N‐1074 has two different isozymes of H‐Lyase, HheA (A‐type) and HheB (B‐type). We have determined their crystal structures to elucidate the differences in enantioselectivity among them. All three groups share a similar structure, including catalytic sites. The lack of enantioselectivity of HheA seems to be due to the relatively wide size of the substrate tunnel compared to that of other H‐Lyases. Among the B‐type H‐Lyases, HheB shows relatively high enantioselectivity compared to that of HheB_GP1. This difference seems to be due to amino acid replacements at the active site tunnel. The binding mode of 1, 3‐dicyano‐2‐propanol at the catalytic site in the crystal structure of the HheB‐DiCN complex suggests that the product should be (R)‐epichlorohydrin, which agrees with the enantioselectivity of HheB. Comparison with the structure of HheC provides a clue for the difference in their enantioselectivity. Proteins 2015; 83:2230–2239. © 2015 Wiley Periodicals, Inc.     

Tim‐2 is the receptor for H‐ferritin on oligodendrocytes

Oligodendrocytes stain more strongly for iron than any other cell in the CNS, and they require iron for the production of myelin. For most cell types transferrin is the major iron delivery protein, yet neither transferrin receptor protein nor mRNA are detectable in mature oligodendrocytes. Thus an alternative iron delivery mechanism must exist. Given the significant long term consequences of developmental iron deficiency and the iron requirements for normal myelination, identification of the iron delivery mechanism for oligodendrocytes is important. Previously we have reported that oligodendrocytes bind H‐ferritin and that H‐ferritin binds to white matter tracts in vivo. Recently, T cell immunoglobulin and mucin domain‐containing protein‐2 (Tim‐2) was shown to bind and internalize H‐ferritin. In the present study we show that Tim‐2 is expressed on oligodendrocytes both in vivo and in vitro. Further, the onset of saturable H‐ferritin binding in CG4 oligodendrocyte cell line is accompanied by Tim‐2 expression. Application of a blocking antibody to the extracellular domain of Tim‐2 significantly reduces H‐ferritin binding to the differentiated CG4 cells and primary oligodendrocytes. Tim‐2 expression on CG4 cells is responsive to iron; decreasing with iron loading and increasing with iron chelation. Taken together, these data provide compelling evidence that Tim‐2 is the H‐ferritin receptor on oligodendrocytes suggesting it is the primary mechanism for iron acquisition by these cells.     

On CO Egress from,and Re‐Uptake by,the Enzyme MauG,as a Mimic of the Acquisition of Oxidizing Agents by the pre‐MADH_MauG System. A Molecular Mechanics Approach

In this work, by applying a non‐deterministic, randomly‐oriented minimal force to the dissociated CO ligand of the MauG‐CO system, the molecular‐dynamics (MD) behavior of this system could be quickly unraveled. It turned out that CO has no marked directional egress from the high‐spin c‐heme iron distal pocket. Rather, CO is able to exploit all interstices created during the protein fluctuations. Nonetheless, no steady route toward the surrounding solvent was ever observed: CO jumped first into other binding pockets before being able to escape the protein. In a few cases, on hitting the surrounding H₂O molecules, CO was observed to reverse direction, re‐entering the protein. A contention that conformational inversion of the P107 ring provides a gate to the iron ion is not supported by the present simulations.     

Comparison of the structures and stabilities of coiled‐coil proteins containing hexafluoroleucine and t‐butylalanine provides insight into the stabilizing effects of highly fluorinated amino acid side‐chains

Highly fluorinated analogs of hydrophobic amino acids are well known to increase the stability of proteins toward thermal unfolding and chemical denaturation, but there is very little data on the structural consequences of fluorination. We have determined the structures and folding energies of three variants of a de novo designed 4‐helix bundle protein whose hydrophobic cores contain either hexafluoroleucine (hFLeu) or t‐butylalanine (tBAla). Although the buried hydrophobic surface area is the same for all three proteins, the incorporation of tBAla causes a rearrangement of the core packing, resulting in the formation of a destabilizing hydrophobic cavity at the center of the protein. In contrast, incorporation of hFLeu, causes no changes in core packing with respect to the structure of the nonfluorinated parent protein which contains only leucine in the core. These results support the idea that fluorinated residues are especially effective at stabilizing proteins because they closely mimic the shape of the natural residues they replace while increasing buried hydrophobic surface area.     

Enantioseparation of Four Organophosphonate Derivatives on N‐(3,5‐Dinitrobenzoyl)‐ l‐leucine–n‐Propylamide Stationary Phase by Molecular Modeling

Four groups of organophosphonate derivatives enantiomers were separated on N‐(3,5‐dinitrobenzoyl)‐S‐leucine chiral stationary phase. The three‐dimensional structures of the complexes between the single enantiotopic chiral compounds and chiral stationary phase have been studied using molecular model and molecular dynamics simulation. Detailed results regarding the conformation, auto‐docking, and thermodynamic estimation are presented. The elution order of the enantiomer could be determined from the energy. The predicted chiral discrimination was obtained by computational results. Chirality 25:101–106, 2013. © 2012 Wiley Periodicals, Inc.