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Michel Guertin - One of the best experts on this subject based on the ideXlab platform.
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Mechanism of the Nitric Oxide Dioxygenase Reaction of Mycobacterium tuberculosis Hemoglobin N
2017Co-Authors: Lavinia Arielle Carabet, Michel Guertin, Patrick Lagüe, Guillaume LamoureuxAbstract:Many globins convert •NO to innocuous NO3– through their nitric oxide dioxygenase (NOD) activity. Mycobacterium tuberculosis fights the oxidative and nitrosative stress imposed by its host (the toxic effects of O2•– and •NO species and their OONO– and •NO2 derivatives) through the action of Truncated Hemoglobin N (trHbN), which catalyzes the NOD reaction with one of the highest rates among globins. The general NOD mechanism comprises the following steps: binding of O2 to the heme, diffusion of •NO into the heme pocket and formation of peroxynitrite (OONO–), isomerization of OONO–, and release of NO3–. Using quantum mechanics/molecular mechanics free-energy calculations, we show that the NOD reaction in trHbN follows a mechanism in which heme-bound OONO– undergoes homolytic cleavage to give FeIVO2– and the •NO2 radical but that these potentially harmful intermediates are short-lived and caged by the heme pocket residues. In particular, the simulations show that Tyr33(B10) side chain is shielded from FeIVO2– and •NO2 (and protected from irreversible oxidation and nitration) by forming stable hydrogen bonds with Gln58(E11) side chain and Leu54(E7) backbone. Aromatic residues Phe46(CD1), Phe32(B9), and Tyr33(B10) promote NO3– dissociation via C–H···O bonding and provide stabilizing interactions for the anion along its egress route
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Ultrafast heme-ligand recombination in Truncated Hemoglobin HbO from Mycobacterium tuberculosis: A ligand cage
Chemical Physics, 2012Co-Authors: Audrius Jasaitis, Michel Guertin, Hugues Ouellet, Jean-christophe Lambry, Jean-louis Martin, Joel Friedman, Marten VosAbstract:Truncated Hemoglobin HbO from Mycobacterium tuberculosis displays very slow exchange of diatomic ligands with its environment. Using femtosecond spectroscopy, we show that upon photoexcitation, ligands rebind with unusual speed and efficiency. Only ∼1% O2 can escape from the heme pocket and less than 1% NO. Most remarkably, CO rebinding occurs for 95%, predominantly in 1.2 ns. The general CO rebinding properties are unexpectedly robust against changes in the interactions with close by aromatic residues Trp88 (G8) and Tyr36 (CD1). Molecular dynamics simulations of the CO complex suggest that interactions of the ligand with structural water molecules as well as its rotational freedom play a role in the high reactivity of the ligand and the heme. The slow exchange of ligands between heme and environment may result from a combination of hindered ligand access to the heme pocket by the network of distal aromatic residues, and low escape probability from the pocket.
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study of the interactions between model membranes and a Truncated Hemoglobin trhbn by nmr and infrared spectroscopy
Biophysical Journal, 2012Co-Authors: Eve Gagne, Michel Guertin, Patrick Lagüe, Michele AugerAbstract:Approximately one third of the world population is infected by the pathogenic bacterium Mycobacterium tuberculosis. A key to the resilience of M. tuberculosis resides in part in its capacity to enter a latent state where it can resist different oxygen and nitrogen oxidative species such as hydrogen peroxide, nitric oxide and peroxynitrite produced by the infected macrophages. One protein responsible for that is trHbN, a Truncated Hemoglobin, that detoxifies nitric oxide from the cellular environment. It is thought that the protein heme achieves that through this mechanism (Mishra et al, J. Am Chem. Soc., 132, 2968-2982):Fe(II)-O2 + NO∗ --> Fe(III) + NO3-The importance of studying this protein lies in the fact that we now have to deal with new antibiotic-resistant strains. Therefore, we have investigated the trHbN orientation and conformation in different lipid model membranes. We also have studied the effect of the protein on these membranes. FTIR was used to observe changes in the conformational order of the lipids in the presence of the protein and the protein secondary structure. Furthermore, solid-state NMR provided information on the membrane conformation and on the protein orientation. These studies were performed in pure lipids, and also in a mixture of two different lipids (TOCL and DOPE) which was optimized to achieve a composition similar to that of the bacterial membrane.
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Structure and Dynamics of Mycobacterium tuberculosis Truncated Hemoglobin N: Insights from NMR Spectroscopy and Molecular Dynamics Simulations
Biochemistry, 2011Co-Authors: Pierre Yves Savard, Michel Guertin, Richard Daigle, Sébastien Morin, Anne Sebilo, Fanny Meindre, Patrick Lagüe, Stéphane GagnéAbstract:The potent nitric oxide dioxygenase (NOD) activity (trHbN-Fe²⁺-O₂ + (•)NO → trHbN-Fe³⁺-OH₂ + NO₃⁻) of Mycobacterium tuberculosis Truncated Hemoglobin N (trHbN) protects aerobic respiration from inhibition by (•)NO. The high activity of trHbN has been attributed in part to the presence of numerous short-lived hydrophobic cavities that allow partition and diffusion of the gaseous substrates (•)NO and O₂ to the active site. We investigated the relation between these cavities and the dynamics of the protein using solution NMR spectroscopy and molecular dynamics (MD). Results from both approaches indicate that the protein is mainly rigid with very limited motions of the backbone N-H bond vectors on the picoseconds-nanoseconds time scale, indicating that substrate diffusion and partition within trHbN may be controlled by side-chains movements. Model-free analysis also revealed the presence of slow motions (microseconds-milliseconds), not observed in MD simulations, for many residues located in helices B and G including the distal heme pocket Tyr33(B10). All currently known crystal structures and molecular dynamics data of Truncated Hemoglobins with the so-called pre-A N-terminal extension suggest a stable α-helical conformation that extends in solution. Moreover, a recent study attributed a crucial role to the pre-A helix for NOD activity. However, solution NMR data clearly show that in near-physiological conditions these residues do not adopt an α-helical conformation and are significantly disordered and that the helical conformation seen in crystal structures is likely induced by crystal contacts. Although this lack of order for the pre-A does not disagree with an important functional role for these residues, our data show that one should not assume an helical conformation for these residues in any functional interpretation. Moreover, future molecular dynamics simulations should not use an initial α-helical conformation for these residues in order to avoid a bias based on an erroneous initial structure for the N-termini residues. This work constitutes the first study of a Truncated Hemoglobin dynamics performed by solution heteronuclear relaxation NMR spectroscopy.
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theoretical investigation of interactions between the Truncated Hemoglobin n from mycobacterium tuberculosis and biological membranes
Biophysical Journal, 2011Co-Authors: Julieanne Rousseau, Michel Guertin, Patrick LagüeAbstract:The Truncated Hemoglobin HbN (trHbN) from the pathogenic bacterium Mycobacterium tuberculosis has a potent ability to detoxify •NO to nitrate (nitric oxide dioxygenase (NOD reaction)) and to protect aerobic respiration from inhibition by •NO in stationary phase cells of M. bovis BCG. HbN catalyses the rapid oxidation of •NO to innocuous nitrate (HbN-FeII(O2) + •NO –> HbN-FeIII + NO3-), with a second-order rate constant k′NOD approximately 745 μM−1 s−1 (23 °C). This protein is thought to play pivotal roles in the cellular metabolism of the bacterium during stress and hypoxia and thus in the persistence of mycobacterial infection. Crystallographic studies and molecular dynamics (MD) simulations revealed that trHbN contains hydrophobic cavities forming four tunnels termed ST, LT, EHT and BET tunnels connecting the active site to the solvant. Recent studies have confirmed that apolar ligands use the tunnels to reach the buried active site from the bulk solvent (Daigle et al., submitted).Until now, trHbN characterization has been done in solution despite the fact that the apolar substrates are more soluble (∼ 3-fold) in biological membranes than in bulk solvent. In this study, we present the results obtained from molecular modelling tools of the interactions of trHbN with model membranes. Preliminary results using implicit solvent suggest that trHbN inserts into the membrane so that the ST and EHT openings are located in the center of the membrane, where the concentration of apolar substrates is the highest and that LT opening is located at the membrane interface. We propose that such positioning may be related to the role of the protein. More results from all-atoms MD simulations will be presented.
Darío A. Estrin - One of the best experts on this subject based on the ideXlab platform.
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Cold-Adaptation Signatures in the Ligand Rebinding Kinetics to the Truncated Hemoglobin of the Antarctic Bacterium Pseudoalteromonas haloplanktis TAC125.
The journal of physical chemistry. B, 2018Co-Authors: Fernando Martín Boubeta, Daniela Giordano, Cinzia Verde, Leonardo Boechi, Darío A. Estrin, Barbara Patrizi, Mariangela Di Donato, Alessandro Iagatti, Stefano Bruno, Stefania AbbruzzettiAbstract:Cold-adapted organisms have evolved proteins endowed with higher flexibility and lower stability in comparison to their thermophilic homologues, resulting in enhanced reaction rates at low temperatures. In this context, protein-bound water molecules were suggested to play a major role, and their weaker interactions at protein active sites have been associated with cold adaptation. In this work, we tested this hypothesis on Truncated Hemoglobins (a family of microbial heme-proteins of yet-unclear function) applying molecular dynamics simulations and ligand-rebinding kinetics on a protein from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 in comparison with its thermophilic Thermobifida fusca homologue. The CO rebinding kinetics of the former highlight several geminate phases, with an unusually long-lived geminate intermediate. An articulated tunnel with at least two distinct docking sites was identified by analysis of molecular dynamics simulations and was suggested to be at the origin of t...
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evolutionary and functional relationships in the Truncated Hemoglobin family
PLOS Computational Biology, 2016Co-Authors: Juan Pablo Bustamante, Leonardo Boechi, Darío A. Estrin, Leandro G Radusky, Arjen Ten Have, Marcelo A. MartíAbstract:Predicting function from sequence is an important goal in current biological research, and although, broad functional assignment is possible when a protein is assigned to a family, predicting functional specificity with accuracy is not straightforward. If function is provided by key structural properties and the relevant properties can be computed using the sequence as the starting point, it should in principle be possible to predict function in detail. The Truncated Hemoglobin family presents an interesting benchmark study due to their ubiquity, sequence diversity in the context of a conserved fold and the number of characterized members. Their functions are tightly related to O2 affinity and reactivity, as determined by the association and dissociation rate constants, both of which can be predicted and analyzed using in-silico based tools. In the present work we have applied a strategy, which combines homology modeling with molecular based energy calculations, to predict and analyze function of all known Truncated Hemoglobins in an evolutionary context. Our results show that Truncated Hemoglobins present conserved family features, but that its structure is flexible enough to allow the switch from high to low affinity in a few evolutionary steps. Most proteins display moderate to high oxygen affinities and multiple ligand migration paths, which, besides some minor trends, show heterogeneous distributions throughout the phylogenetic tree, again suggesting fast functional adaptation. Our data not only deepens our comprehension of the structural basis governing ligand affinity, but they also highlight some interesting functional evolutionary trends.
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Following Ligand Migration Pathways from Picoseconds to Milliseconds in Type II Truncated Hemoglobin from
2015Co-Authors: Thermobifida Fusca, Stefania Abbruzzetti, Alberto Boffi, Juan Pablo Bustamante, Agnese Marcelli, Ro Feis, Ra Bonamore, Cristina Gellini, Pier Remigio Salvi, Darío A. EstrinAbstract:CO recombination kinetics has been investigated in the type II Truncated Hemoglobin from Thermobifida fusca (Tf-trHb) over more than 10 time decades (from 1 ps to,100 ms) by combining femtosecond transient absorption, nanosecond laser flash photolysis and optoacoustic spectroscopy. Photolysis is followed by a rapid geminate recombination with a time constant of,2 ns representing almost 60 % of the overall reaction. An additional, small amplitude geminate recombination was identified at,100 ns. Finally, CO pressure dependent measurements brought out the presence of two transient species in the second order rebinding phase, with time constants ranging from,3 to,100 ms. The available experimental evidence suggests that the two transients are due to the presence of two conformations which do not interconvert within the time frame of the experiment. Computational studies revealed that the plasticity of protein structure is able to define a branched pathway connecting the ligand binding site and the solvent. This allowed to build a kinetic model capable o
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Structural Flexibility of the Heme Cavity in the Cold-Adapted Truncated Hemoglobin from the Antarctic Marine Bacterium Pseudoalteromonas Haloplanktis Tac125.
The FEBS journal, 2015Co-Authors: Daniela Giordano, Alessandra Pesce, Guido Di Prisco, Alessia Riccio, Barry D. Howes, Leonardo Boechi, E. Caldelli, Marco Nardini, Juan Pablo Bustamante, Darío A. EstrinAbstract:Truncated Hemoglobins build one of the three branches of the globin protein superfamily. They display a characteristic two-on-two α-helical sandwich fold and are clustered into three groups (I, II and III) based on distinct structural features. Truncated Hemoglobins are present in eubacteria, cyanobacteria, protozoa and plants. Here we present a structural, spectroscopic and molecular dynamics characterization of a group-II Truncated Hemoglobin, encoded by the PSHAa0030 gene from Pseudoalteromonas haloplanktis TAC125 (Ph-2/2HbO), a cold-adapted Antarctic marine bacterium hosting one flavoHemoglobin and three distinct Truncated Hemoglobins. The Ph-2/2HbO aquo-met crystal structure (at 2.21 A resolution) shows typical features of group-II Truncated Hemoglobins, namely the two-on-two α-helical sandwich fold, a helix Φ preceding the proximal helix F, and a heme distal-site hydrogen-bonded network that includes water molecules and several distal-site residues, including His(58)CD1. Analysis of Ph-2/2HbO by electron paramagnetic resonance, resonance Raman and electronic absorption spectra, under varied solution conditions, shows that Ph-2/2HbO can access diverse heme ligation states. Among these, detection of a low-spin heme hexa-coordinated species suggests that residue Tyr(42)B10 can undergo large conformational changes in order to act as the sixth heme-Fe ligand. Altogether, the results show that Ph-2/2HbO maintains the general structural features of group-II Truncated Hemoglobins but displays enhanced conformational flexibility in the proximity of the heme cavity, a property probably related to the functional challenges, such as low temperature, high O2 concentration and low kinetic energy of molecules, experienced by organisms living in the Antarctic environment. Database Structural data have been submitted to the Protein Data Bank under accession numbers 4UUR and R4UURSF
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Structural flexibility of the heme cavity in the cold-adapted Truncated Hemoglobin from the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125
'Wiley', 2015Co-Authors: Daniela Giordano, Guido Di Prisco, Alessia Riccio, Barry D. Howes, Leonardo Boechi, A. Pesce, J.p. Bustamante, E. Caldelli, M. Nardini, Darío A. EstrinAbstract:Truncated Hemoglobins build one of the three branches of the globin protein superfamily. They display a characteristic two-on-two \u3b1-helical sandwich fold and are clustered into three groups (I, II and III) based on distinct structural features. Truncated Hemoglobins are present in eubacteria, cyanobacteria, protozoa and plants. Here we present a structural, spectroscopic and molecular dynamics characterization of a group-II Truncated Hemoglobin, encoded by the PSHAa0030 gene from Pseudoalteromonas haloplanktis TAC125 (Ph-2/2HbO), a cold-adapted Antarctic marine bacterium hosting one flavoHemoglobin and three distinct Truncated Hemoglobins. The Ph-2/2HbO aquo-met crystal structure (at 2.21 \uc5 resolution) shows typical features of group-II Truncated Hemoglobins, namely the two-on-two \u3b1-helical sandwich fold, a helix \u3a6 preceding the proximal helix F, and a heme distal-site hydrogen-bonded network that includes water molecules and several distal-site residues, including His(58)CD1. Analysis of Ph-2/2HbO by electron paramagnetic resonance, resonance Raman and electronic absorption spectra, under varied solution conditions, shows that Ph-2/2HbO can access diverse heme ligation states. Among these, detection of a low-spin heme hexa-coordinated species suggests that residue Tyr(42)B10 can undergo large conformational changes in order to act as the sixth heme-Fe ligand. Altogether, the results show that Ph-2/2HbO maintains the general structural features of group-II Truncated Hemoglobins but displays enhanced conformational flexibility in the proximity of the heme cavity, a property probably related to the functional challenges, such as low temperature, high O2 concentration and low kinetic energy of molecules, experienced by organisms living in the Antarctic environment
Martino Bolognesi - One of the best experts on this subject based on the ideXlab platform.
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structural determinants of ligand migration in mycobacterium tuberculosis Truncated Hemoglobin o
Proteins, 2008Co-Authors: Leonardo Boechi, Martino Bolognesi, Marcelo A. Martí, Mario Milani, Javier F Luque, Darío A. EstrinAbstract:Mycobacterium tuberculosis is the causative agent of human tuberculosis, one of the most prevalent infectious diseases in the world. Its genome hosts the glbN and glbO genes coding for two proteins, Truncated Hemoglobin N (trHbN) and Truncated Hemoglobin O (trHbO), that belong to different groups (I and II, respectively) of the recently discovered trHb family of hemeproteins. The different expression pattern and kinetics rates constants for ligand association and NO oxidation rate suggest different functions for these proteins. Previous experimental and theoretical studies showed that, in trHbs, ligand migration along the internal tunnel cavity system is a key issue in determining the ligand-binding characteristics. The X-ray structure of trHbO has been solved and shows several internal cavities and secondary-docking sites. In this work, we present an extensive investigation of the tunnel/cavity system ofM. tuberculosis trHbO by means of computer-simulation techniques. We have computed the free-energy profiles for ligand migration along three found tunnels in the oxy and deoxy w.t. and mutant trHbO proteins. Our results show that multiple-ligand migration paths are possible and that several conserved residues such as TrpG8 play a key role in the ligand-migration regulation. Proteins 2008. © 2008 Wiley-Liss, Inc.
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ligand binding to Truncated Hemoglobin n from mycobacterium tuberculosis is strongly modulated by the interplay between the distal heme pocket residues and internal water
Journal of Biological Chemistry, 2008Co-Authors: Yannick Ouellet, Martino Bolognesi, Mario Milani, Richard Daigle, Patrick Lagüe, David Dantsker, Joel M Friedman, Michel GuertinAbstract:Abstract The survival of Mycobacterium tuberculosis requires detoxification of host ·NO. Oxygenated Mycobacterium tuberculosis Truncated Hemoglobin N catalyzes the rapid oxidation of nitric oxide to innocuous nitrate with a second-order rate constant ( ≈ 745 × 106 m-1·s-1), which is ∼15-fold faster than the reaction of horse heart myoglobin. We ask what aspects of structure and/or dynamics give rise to this enhanced reactivity. A first step is to expose what controls ligand/substrate binding to the heme. We present evidence that the main barrier to ligand binding to deoxy-Truncated Hemoglobin N (deoxy-trHbN) is the displacement of a distal cavity water molecule, which is mainly stabilized by residue Tyr(B10) but not coordinated to the heme iron. As observed in the Tyr(B10)/Gln(E11) apolar mutants, once this kinetic barrier is lowered, CO and O2 binding is very rapid with rates approaching 1-2 × 109 m-1·s-1. These large values almost certainly represent the upper limit for ligand binding to a heme protein and also indicate that the iron atom in trHbN is highly reactive. Kinetic measurements on the photoproduct of the ·NO derivative of met-trHbN, where both the ·NO and water can be directly followed, revealed that water rebinding is quite fast (∼1.49 × 108 s-1) and is responsible for the low geminate yield in trHbN. Molecular dynamics simulations, performed with trHbN and its distal mutants, indicated that in the absence of a distal water molecule, ligand access to the heme iron is not hindered. They also showed that a water molecule is stabilized next to the heme iron through hydrogen-bonding with Tyr(B10) and Gln(E11).
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ferrous campylobacter jejuni Truncated Hemoglobin p displays an extremely high reactivity for cyanide a comparative study
FEBS Journal, 2008Co-Authors: Alessandro Bolli, Paolo Visca, Alessandra Pesce, Martino Bolognesi, M. Nardini, Michel Guertin, Chiara Ciaccio, Massimo Coletta, Paolo AscenziAbstract:Campylobacter jejuni hosts two Hemoglobins (Hbs). The Camplylobacter jejuni single-domain Hb (called Cgb) is homologous to the globin domain of flavoHemoglobin, and it has been proposed to protect the bacterium against nitrosative stress. The second Hb is called Ctb (hereafter Cj-trHbP), belongs to Truncated Hb group III, and has been hypothesized to be involved in O2 chemistry. Here, the kinetics and thermodynamics of cyanide binding to ferric and ferrous Cj-trHbP [Cj-trHbP(III) and Cj-trHbP(II), respectively] are reported and analyzed in parallel with those of related heme proteins, with particular reference to those from Mycobacterium tuberculosis. The affinity of cyanide for Cj-trHbP(II) is higher than that reported for any known (in)vertebrate globin by more than three orders of magnitude (K = 1.2 × 10−6 m). This can be fully attributed to the highest (ever observed for a ferrous Hb) cyanide-binding association rate constant (kon = 3.3 × 103 m−1·s−1), even though the binding process displays a rate-limiting step (kmax = 9.1 s−1). Cj-trHbP(III) shows a very high affinity for cyanide (L = 5.8 × 10−9 m); however, cyanide association kinetics are independent of cyanide concentration, displaying a rate-limiting step (lmax = 2.0 × 10−3 s−1). Values of the first-order rate constant for cyanide dissociation from Cj-trHbP(II)–cyanide and Cj-trHbP(III)–cyanide (koff =5.0 × 10−3 s−1 and loff ≥ 1 × 10−4 s−1, respectively) are similar to those reported for (in)vertebrate globins. The very high affinity of cyanide for Cj-trHbP(II), reminiscent of that of horseradish peroxidase(II), suggests that this globin may participate in cyanide detoxification.
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the roles of tyr cd1 and trp g8 in mycobacterium tuberculosis Truncated Hemoglobin o in ligand binding and on the heme distal site architecture
Biochemistry, 2007Co-Authors: Hugues Ouellet, Martino Bolognesi, Manon Couture, Marie Labarre, Mario Milani, Michel GuertinAbstract:The crystal structure of the cyano-met form of Mt-trHbO revealed two unusual distal residues Y(CD1) and W(G8) forming a hydrogen-bond network with the heme-bound ligand [Milani, M., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 5766−5771]. W(G8) is an invariant residue in group II and group III trHbs and has no counterpart in other globins. A previous study reported that changing Y(CD1) for a Phe causes a significant increase in the O2 combination rate, but almost no change in the O2 dissociation rate [Ouellet, H., et al. (2003) Biochemistry 42, 5764−5774]. Here we investigated the role of the W(G8) in ligand binding by using resonance Raman spectroscopy, stopped-flow spectrophotometry, and X-ray crystallography. For this purpose, W(G8) was changed, by site-directed mutagenesis, to a Phe in both the wild-type protein and the mutant Y(CD1)F to create the single mutant W(G8)F and the double mutant Y(CD1)F/W(G8)F, respectively. Resonance Raman results suggest that W(G8) interacts with the heme-bound O2 an...
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protein fold and structure in the Truncated 2 2 globin family
Gene, 2007Co-Authors: M. Nardini, Alessandra Pesce, Mario Milani, Martino BolognesiAbstract:Abstract Analysis of amino acids sequences and protein folds has recently unraveled the structural bases and details of several proteins from the recently discovered “Truncated Hemoglobin” family. The analysis here presented, in agreement with previous surveys, shows that Truncated Hemoglobins can be classified in three main groups, based on their structural properties. Crystallographic analyses have shown that all three groups adopt a 2-on-2 α-helical sandwich fold, resulting from apparent editing of the classical 3-on-3 α-helical sandwich of vertebrate and invertebrate conventional globins. Specific structural features distinguish each of the three groups. Among these, a protein matrix tunnel system is typical of group I, a Trp residue at the G8 topological site is conserved in groups II and III, and TyrB10 is almost invariant through the three groups. A strongly intertwined network of hydrogen bonds stabilizes the heme bound ligand, despite variability of the heme distal residues observed in the different proteins considered. Details of ligand recognition in the three groups are discussed at the light of residue conservation and of differing ligand diffusion pathways to the heme. Based on structural analyses of the family-specific fold, we endorse a recent proposal of leaving the “Truncated Hemoglobins” term, that does not represent properly the observed 2-on-2 α-helical sandwich fold, and adopting the simple “2/2Hb” term to concisely address this protein family.
Markus Meuwly - One of the best experts on this subject based on the ideXlab platform.
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kinetic analysis and structural interpretation of competitive ligand binding for no dioxygenation in Truncated Hemoglobin n
Angewandte Chemie, 2018Co-Authors: Akshaya K Das, Markus MeuwlyAbstract:The conversion of nitric oxide (NO) into nitrate (NO3- ) by dioxygenation protects cells from lethal NO. Starting from NO-bound heme, the first step in converting NO into benign NO3- is the ligand exchange reaction FeNO+O2 →FeO2 +NO, which is still poorly understood at a molecular level. For wild-type (WT) Truncated Hemoglobin N (trHbN) and its Y33A mutant, the calculated barriers for the exchange reaction differ by 1.5 kcal mol-1 , compared with 1.7 kcal mol-1 from experiment. It is directly confirmed that the ligand exchange reaction is rate-limiting in trHbN and that entropic contributions account for 75 % of the difference between the WT and the mutant. Residues Tyr 33, Phe 46, Val 80, His 81, and Gln 82 surrounding the active site are expected to control the reaction path. By comparison with electronic structure calculations, the transition state separating the two ligand-bound states was assigned to a 2 A state.
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migration of small ligands in globins xe diffusion in Truncated Hemoglobin n
PLOS Computational Biology, 2017Co-Authors: Polydefkis Diamantis, Oliver T Unke, Markus MeuwlyAbstract:In heme proteins, the efficient transport of ligands such as NO or O2 to the binding site is achieved via ligand migration networks. A quantitative assessment of ligand diffusion in these networks is thus essential for a better understanding of the function of these proteins. For this, Xe migration in Truncated Hemoglobin N (trHbN) of Mycobacterium Tuberculosis was studied using molecular dynamics simulations. Transitions between pockets of the migration network and intra-pocket relaxation occur on similar time scales (10 ps and 20 ps), consistent with low free energy barriers (1-2 kcal/mol). Depending on the pocket from where Xe enters a particular transition, the conformation of the side chains lining the transition region differs which highlights the coupling between ligand and protein degrees of freedom. Furthermore, comparison of transition probabilities shows that Xe migration in trHbN is a non-Markovian process. Memory effects arise due to protein rearrangements and coupled dynamics as Xe moves through it.
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coupled protein ligand dynamics in Truncated Hemoglobin n from atomistic simulations and transition networks
Biochimica et Biophysica Acta, 2015Co-Authors: Pierreandre Cazade, Markus Meuwly, Ganna BerezovskaAbstract:Abstract Background The nature of ligand motion in proteins is difficult to characterize directly usingexperiment. Specifically, it is unclear to what degree these motions are coupled. Methods All-atom simulations are used to sample ligand motion in Truncated Hemoglobin N. A transition network analysis including ligand- and protein-degrees of freedom is used to analyze the microscopic dynamics. Results Clustering of two different subsets of MD trajectories highlights the importance of a diverse and exhaustive description to define the macrostates for a ligand-migration network. Monte Carlo simulations on the transition matrices from one particular clustering are able to faithfully capture the atomistic simulations. Contrary to clustering by ligand positions only, including a protein degree of freedom yields considerably improved coarse grained dynamics. Analysis with and without imposing detailed balance agree closely which suggests that the underlying atomistic simulations are converged with respect to sampling transitions between neighboring sites. Conclusions Protein and ligand dynamics are not independent from each other and ligand migration through globular proteins is not passive diffusion. General significance Transition network analysis is a powerful tool to analyze and characterize the microscopic dynamics in complex systems. This article is part of a Special Issue entitled Recent developments of molecular dynamics.
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a comparative analysis of clustering algorithms o2 migration in Truncated Hemoglobin i from transition networks
Journal of Chemical Physics, 2015Co-Authors: Pierreandre Cazade, Ganna Berezovska, Wenwei Zheng, Diego Pradagracia, Francesco Rao, Cecilia Clementi, Markus MeuwlyAbstract:The ligand migration network for O2–diffusion in Truncated Hemoglobin N is analyzed based on three different clustering schemes. For coordinate-based clustering, the conventional k–means and the kinetics-based Markov Clustering (MCL) methods are employed, whereas the locally scaled diffusion map (LSDMap) method is a collective-variable-based approach. It is found that all three methods agree well in their geometrical definition of the most important docking site, and all experimentally known docking sites are recovered by all three methods. Also, for most of the states, their population coincides quite favourably, whereas the kinetics of and between the states differs. One of the major differences between k–means and MCL clustering on the one hand and LSDMap on the other is that the latter finds one large primary cluster containing the Xe1a, IS1, and ENT states. This is related to the fact that the motion within the state occurs on similar time scales, whereas structurally the state is found to be quite diverse. In agreement with previous explicit atomistic simulations, the Xe3 pocket is found to be a highly dynamical site which points to its potential role as a hub in the network. This is also highlighted in the fact that LSDMap cannot identify this state. First passage time distributions from MCL clusterings using a one- (ligand-position) and two-dimensional (ligand-position and protein-structure) descriptor suggest that ligand- and protein-motions are coupled. The benefits and drawbacks of the three methods are discussed in a comparative fashion and highlight that depending on the questions at hand the best-performing method for a particular data set may differ.
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oxygen migration pathways in no bound Truncated Hemoglobin
ChemPhysChem, 2012Co-Authors: Pierreandre Cazade, Markus MeuwlyAbstract:Atomistic simulations of dioxygen (O(2)) dynamics and migration in nitric oxide-bound Truncated Hemoglobin N (trHbN) of Mycobacterium tuberculosis are reported. From more than 100 ns of simulations the connectivity network involving the metastable states for localization of the O(2) ligand is built and analyzed. It is found that channel I is the primary entrance point for O(2) whereas channel II is predominantly an exit path although access to the protein active site is also possible. For O(2) a new site compared to nitric oxide, from which reaction with the heme group can occur, was found. As this site is close to the heme iron, it could play an important role in the dioxygenation mechanism as O(2) can remain there for hundreds of picoseconds after which it can eventually leave the protein, while NO is localized in Xe2. The present study supports recent experimental work which proposed that O(2) docks in alternative pockets than Xe close to the reactive site. Similar to other proteins, a phenylalanine residue (Phe62) plays the role of a gate along the access route in channel I. The most highly connected site is the Xe3 pocket which is a "hub" and free energy barriers between the different metastable states are ≈1.5 kcal mol(-1) which allows facile O(2) migration within the protein.
Mario Milani - One of the best experts on this subject based on the ideXlab platform.
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structural determinants of ligand migration in mycobacterium tuberculosis Truncated Hemoglobin o
Proteins, 2008Co-Authors: Leonardo Boechi, Martino Bolognesi, Marcelo A. Martí, Mario Milani, Javier F Luque, Darío A. EstrinAbstract:Mycobacterium tuberculosis is the causative agent of human tuberculosis, one of the most prevalent infectious diseases in the world. Its genome hosts the glbN and glbO genes coding for two proteins, Truncated Hemoglobin N (trHbN) and Truncated Hemoglobin O (trHbO), that belong to different groups (I and II, respectively) of the recently discovered trHb family of hemeproteins. The different expression pattern and kinetics rates constants for ligand association and NO oxidation rate suggest different functions for these proteins. Previous experimental and theoretical studies showed that, in trHbs, ligand migration along the internal tunnel cavity system is a key issue in determining the ligand-binding characteristics. The X-ray structure of trHbO has been solved and shows several internal cavities and secondary-docking sites. In this work, we present an extensive investigation of the tunnel/cavity system ofM. tuberculosis trHbO by means of computer-simulation techniques. We have computed the free-energy profiles for ligand migration along three found tunnels in the oxy and deoxy w.t. and mutant trHbO proteins. Our results show that multiple-ligand migration paths are possible and that several conserved residues such as TrpG8 play a key role in the ligand-migration regulation. Proteins 2008. © 2008 Wiley-Liss, Inc.
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ligand binding to Truncated Hemoglobin n from mycobacterium tuberculosis is strongly modulated by the interplay between the distal heme pocket residues and internal water
Journal of Biological Chemistry, 2008Co-Authors: Yannick Ouellet, Martino Bolognesi, Mario Milani, Richard Daigle, Patrick Lagüe, David Dantsker, Joel M Friedman, Michel GuertinAbstract:Abstract The survival of Mycobacterium tuberculosis requires detoxification of host ·NO. Oxygenated Mycobacterium tuberculosis Truncated Hemoglobin N catalyzes the rapid oxidation of nitric oxide to innocuous nitrate with a second-order rate constant ( ≈ 745 × 106 m-1·s-1), which is ∼15-fold faster than the reaction of horse heart myoglobin. We ask what aspects of structure and/or dynamics give rise to this enhanced reactivity. A first step is to expose what controls ligand/substrate binding to the heme. We present evidence that the main barrier to ligand binding to deoxy-Truncated Hemoglobin N (deoxy-trHbN) is the displacement of a distal cavity water molecule, which is mainly stabilized by residue Tyr(B10) but not coordinated to the heme iron. As observed in the Tyr(B10)/Gln(E11) apolar mutants, once this kinetic barrier is lowered, CO and O2 binding is very rapid with rates approaching 1-2 × 109 m-1·s-1. These large values almost certainly represent the upper limit for ligand binding to a heme protein and also indicate that the iron atom in trHbN is highly reactive. Kinetic measurements on the photoproduct of the ·NO derivative of met-trHbN, where both the ·NO and water can be directly followed, revealed that water rebinding is quite fast (∼1.49 × 108 s-1) and is responsible for the low geminate yield in trHbN. Molecular dynamics simulations, performed with trHbN and its distal mutants, indicated that in the absence of a distal water molecule, ligand access to the heme iron is not hindered. They also showed that a water molecule is stabilized next to the heme iron through hydrogen-bonding with Tyr(B10) and Gln(E11).
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the roles of tyr cd1 and trp g8 in mycobacterium tuberculosis Truncated Hemoglobin o in ligand binding and on the heme distal site architecture
Biochemistry, 2007Co-Authors: Hugues Ouellet, Martino Bolognesi, Manon Couture, Marie Labarre, Mario Milani, Michel GuertinAbstract:The crystal structure of the cyano-met form of Mt-trHbO revealed two unusual distal residues Y(CD1) and W(G8) forming a hydrogen-bond network with the heme-bound ligand [Milani, M., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 5766−5771]. W(G8) is an invariant residue in group II and group III trHbs and has no counterpart in other globins. A previous study reported that changing Y(CD1) for a Phe causes a significant increase in the O2 combination rate, but almost no change in the O2 dissociation rate [Ouellet, H., et al. (2003) Biochemistry 42, 5764−5774]. Here we investigated the role of the W(G8) in ligand binding by using resonance Raman spectroscopy, stopped-flow spectrophotometry, and X-ray crystallography. For this purpose, W(G8) was changed, by site-directed mutagenesis, to a Phe in both the wild-type protein and the mutant Y(CD1)F to create the single mutant W(G8)F and the double mutant Y(CD1)F/W(G8)F, respectively. Resonance Raman results suggest that W(G8) interacts with the heme-bound O2 an...
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protein fold and structure in the Truncated 2 2 globin family
Gene, 2007Co-Authors: M. Nardini, Alessandra Pesce, Mario Milani, Martino BolognesiAbstract:Abstract Analysis of amino acids sequences and protein folds has recently unraveled the structural bases and details of several proteins from the recently discovered “Truncated Hemoglobin” family. The analysis here presented, in agreement with previous surveys, shows that Truncated Hemoglobins can be classified in three main groups, based on their structural properties. Crystallographic analyses have shown that all three groups adopt a 2-on-2 α-helical sandwich fold, resulting from apparent editing of the classical 3-on-3 α-helical sandwich of vertebrate and invertebrate conventional globins. Specific structural features distinguish each of the three groups. Among these, a protein matrix tunnel system is typical of group I, a Trp residue at the G8 topological site is conserved in groups II and III, and TyrB10 is almost invariant through the three groups. A strongly intertwined network of hydrogen bonds stabilizes the heme bound ligand, despite variability of the heme distal residues observed in the different proteins considered. Details of ligand recognition in the three groups are discussed at the light of residue conservation and of differing ligand diffusion pathways to the heme. Based on structural analyses of the family-specific fold, we endorse a recent proposal of leaving the “Truncated Hemoglobins” term, that does not represent properly the observed 2-on-2 α-helical sandwich fold, and adopting the simple “2/2Hb” term to concisely address this protein family.
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protein structure in the Truncated 2 2 Hemoglobin family
Iubmb Life, 2007Co-Authors: Alessandra Pesce, M. Nardini, Mario Milani, Martino BolognesiAbstract:Summary The discovery of protein sequences belonging to the widespread ‘Truncated Hemoglobin’ family has been followed in the last few years by extensive analyses of their three-dimensional structures. Truncated Hemoglobins can be classified in three main groups, in light of their overall structural properties. The three groups adopt a 2-on-2 a-helical sandwich fold, based on four main a-helices of the classical 3-on-3 a-helical sandwich found in vertebrate and invertebrate globins. Each of the three groups displays sequence and structure specific features. Among these, a protein matrix tunnel system is typical of group I, a Trp residue at the G8 topological site is conserved in groups II and III, and residue TyrB10 is almost invariant in the three groups. Despite sequence variability in the heme distal site region, a strongly intertwined, but varied, network of hydrogen bonds stabilizes the heme ligand in the three protein groups. Fine mechanisms of ligand recognition and stabilization may vary based on groupspecific distal site residues and on differing ligand diffusion pathways to the heme. Taken together, the structural considerations here presented underline that ‘Truncated Hemoglobins’ result from careful editing of the 3-on-3 a-helical globin sandwich fold, rather than from simple ‘truncation’ events. Thus, ‘2/2Hb’ appears the most proper term to concisely address this protein family. IUBMB Life, 59: 535–541, 2007
