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The influence of pressure on the equilibrium between five-(5c) and six-coordination (6c) forms in neuroglobin (Ngb) and myoglobin (Mb) has been examined by means of molecular dynamics (MD) simulations at normal and high pressure. The results show that the main effect of high pressure is to reduce the protein mobility without altering the structure in a significant manner. Moreover, our data suggest that the equilibrium between 5c and 6c states in globins is largely controlled by the structure and dynamics of the C-D region. Finally, in agreement with the available experimental data, the free energy profiles obtained from steered MD for both proteins indicate that high pressure enhances hexacoordination. In Ngb, the shift in equilibrium is mainly related to an increase in the 6c→5c transition barrier, whereas in Mb such a shift is primarily due to a destabilization of the 5c state. Proteins 2009. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Nitrobindins (Nbs) are all-β-barrel heme proteins present along the evolutionary ladder. They display a highly solvent-exposed ferric heme group with the iron atom being coordinated by the proximal His residue and a water molecule at the distal position. Ferric nitrobindins (Nb(III)) play a role in the conversion of toxic peroxynitrite (ONOO - ) to harmless nitrate, with the value of the second-order rate constant being similar to those of most heme proteins. The value of the second-order rate constant of Nbs increases as the pH decreases; this suggests that Nb(III) preferentially reacts with peroxynitrous acid (ONOOH), although ONOO - is more nucleophilic. In this work, we shed light on the molecular basis of the ONOO - and ONOOH reactivity of ferric Mycobacterium tuberculosis Nb ( Mt -Nb(III)) by dissecting the ligand migration toward the active site, the water molecule release, and the ligand binding process by computer simulations. Classical molecular dynamics simulations were performed by employing a steered molecular dynamics approach and the Jarzynski equality to obtain ligand migration free energy profiles for both ONOO - and ONOOH. Our results indicate that ONOO - and ONOOH migration is almost unhindered, consistent with the exposed metal center of Mt -Nb(III). To further analyze the ligand binding process, we computed potential energy profiles for the displacement of the Fe(III)-coordinated water molecule using a hybrid QM/MM scheme at the DFT level and a nudged elastic band approach. These results indicate that ONOO - exhibits a much larger barrier for ligand displacement than ONOOH, suggesting that water displacement is assisted by protonation of the leaving group by the incoming ONOOH.

The interactions of the heme iron of hemeproteins with sulfide and disulfide compounds are of potential interest as physiological signaling processes. While the interaction with hydrogen sulfide has been described computationally and experimentally, the reaction with disulfide, and specifically the molecular mechanism for ligand binding has not been studied in detail. In this work, we study the association process for disulfane and its conjugate base disulfanide at different pH conditions. Additionally, by means of advanced sampling techniques based on multiple steered molecular dynamics, we provide free energy profiles for ligand migration for both acid/base species, showing a similar behavior to the previously reported for the related H 2 S/HS¯ pair. Finally, we studied the ligand interchange reaction (H 2 O/H 2 S, HS¯ and H 2 O/HSSH, HSS¯) by means of hybrid quantum mechanics-molecular mechanics calculations. We show that the anionic species are able to displace more efficiently the H 2 O bound to the iron, and that the H-bond network in the distal cavity can help the neutral species to perform the reaction. Altogether, we provide a molecular explanation for the experimental information and show that the global association process depends on a fine balance between the migration towards the active site and the ligand interchange reaction., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The mitochondrial cysteine desulfurase NFS1 is an essential PLP-dependent enzyme involved in iron-sulfur cluster assembly. The enzyme catalyzes the desulfurization of the l-Cys substrate, producing a persulfide and l-Ala as products. In this study, we set the measurement of the product l-Ala by NMR in vitro by means of 1 H NMR spectra acquisition. This methodology provided us with the possibility of monitoring the reaction in both fixed-time and real-time experiments, with high sensitivity and accuracy. By studying I452A, W454A, Q456A, and H457A NFS1 variants, we found that the C-terminal stretch (CTS) of the enzyme is critical for function. Specifically, mutation of the extremely conserved position W454 resulted in highly decreased activity. Additionally, we worked on two singular variants: " GGG " and C158A. In the former, the catalytic Cys-loop was altered by including two Gly residues to increase the flexibility of this loop. This variant had significantly impaired activity, indicating that the Cys-loop motions are fine-tuned in the wild-type enzyme. In turn, for C158A, we found an unanticipated increase in l-Cys desulfurase activity. Furthermore, we carried out molecular dynamics simulations of the supercomplex dedicated to iron-sulfur cluster biosynthesis, which includes NFS1, ACP, ISD11, ISCU2, and FXN subunits. We identified CTS as a key element that established interactions with ISCU2 and FXN concurrently; we found specific interactions that are established when FXN is present, reinforcing the idea that FXN not only forms part of the iron-sulfur cluster assembly site but also modulates the internal motions of ISCU2.

During the second half of the 20th century, following structural biology hallmark works on DNA and proteins, biochemists shifted their questions from "what does this molecule look like?" to "how does this process work?". Prompted by the theoretical and practical developments in computational chemistry, this led to the emergence of biomolecular simulations and, along with the 2013 Nobel Prize in Chemistry, to the development of hybrid QM/MM methods. QM/MM methods are necessary whenever the problem we want to address involves chemical reactivity and/or a change in the system's electronic structure, with archetypal examples being the studies of an enzyme's reaction mechanism and a metalloprotein's active site. In the last decades QM/MM methods have seen an increasing adoption driven by their incorporation in widely used biomolecular simulation software. However, properly setting up a QM/MM simulation is not an easy task, and several issues need to be properly addressed to obtain meaningful results. In the present work, we describe both the theoretical concepts and practical issues that need to be considered when performing QM/MM simulations. We start with a brief historical perspective on the development of these methods and describe when and why QM/MM methods are mandatory. Then we show how to properly select and analyze the performance of the QM level of theory, the QM system size, and the position and type of the boundaries. We show the relevance of performing prior QM model system (or QM cluster) calculations in a vacuum and how to use the corresponding results to adequately calibrate those derived from QM/MM. We also discuss how to prepare the starting structure and how to select an adequate simulation strategy, including those based on geometry optimizations as well as free energy methods. In particular, we focus on the determination of free energy profiles using multiple steered molecular dynamics (MSMD) combined with Jarzynski's equation. Finally, we describe the results for two illustrative and complementary examples: the reaction performed by chorismate mutase and the study of ligand binding to hemoglobins. Overall, we provide many practical recommendations (or shortcuts) together with important conceptualizations that we hope will encourage more and more researchers to incorporate QM/MM studies into their research projects.

Root hair cells are important sensors of soil conditions. They grow towards and absorb water-soluble nutrients. This fast and oscillatory growth is mediated by continuous remodeling of the cell wall. Root hair cell walls contain polysaccharides and hydroxyproline-rich glycoproteins, including extensins (EXTs). Class-III peroxidases (PRXs) are secreted into the apoplastic space and are thought to trigger either cell wall loosening or polymerization of cell wall components, such as Tyr-mediated assembly of EXT networks (EXT-PRXs). The precise role of these EXT-PRXs is unknown. Using genetic, biochemical, and modeling approaches, we identified and characterized three root-hair-specific putative EXT-PRXs, PRX01, PRX44, and PRX73. prx01,44,73 triple mutation and PRX44 and PRX73 overexpression had opposite effects on root hair growth, peroxidase activity, and ROS production, with a clear impact on cell wall thickness. We use an EXT fluorescent reporter with contrasting levels of cell wall insolubilization in prx01,44,73 and PRX44-overexpressing background plants. In this study, we propose that PRX01, PRX44, and PRX73 control EXT-mediated cell wall properties during polar expansion of root hair cells.

In humans, the loss of frataxin results in Friedreich's Ataxia, a neurodegenerative disease, in which a deficit in the iron-sulfur cluster assembly is observed. In this work, we analyzed three frataxin variants in which one tryptophan was replaced by a glycine: W155G, W168G and W173G. As expected, given its localization in the assembly site, W155G was not able to activate the desulfurase activity of the supercomplex for iron-sulfur cluster assembly. In turn, W168G, which was significantly more unstable than W155G, was fully active. W173G, which was highly unstable as W168G, showed a significantly decreased activity, only slightly higher than W155G. As W168G and W173G were highly sensitive to proteolysis, we investigated the protein motions by molecular dynamic simulations. We observed that W173G may display altered motions at the Trp155 site. Furthermore, we revealed a H-bond network in which Trp155 takes part, involving residues Gln148, Asn151, Gln153 and Arg165. We suggest that this motion modulation that specifically alters the population of different Trp155 rotamers can be directly transferred to the assembly site, altering the dynamics of the ISCU His137 key residue. This hypothesis was also contrasted by means of molecular dynamic simulations of frataxin in the context of the complete supercomplex. We propose that the supercomplex requires very definite motions of Trp155 to consolidate the assembly site., (Copyright © 2021 Elsevier Inc. All rights reserved.)

THB1 is a monomeric truncated hemoglobin from the green alga Chlamydomonas reinhardtii. In the absence of exogenous ligands and at neutral pH, the heme group of THB1 is coordinated by two protein residues, Lys53 and His77. THB1 is thought to function as a nitric oxide dioxygenase, and the distal binding of O 2 requires the cleavage of the Fe-Lys53 bond accompanied by protonation and expulsion of the lysine from the heme cavity into the solvent. Nuclear magnetic resonance spectroscopy and crystallographic data have provided dynamic and structural insights of the process, but the details of the mechanism have not been fully elucidated. We applied a combination of computer simulations and site-directed mutagenesis experiments to shed light on this issue. Molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics restrained optimizations were performed to explore the nature of the transition between the decoordinated and lysine-bound states of the ferrous heme in THB1. Lys49 and Arg52, which form ionic interactions with the heme propionates in the X-ray structure of lysine-bound THB1, were observed to assist in maintaining Lys53 inside the protein cavity and play a key role in the transition. Lys49Ala, Arg52Ala and Lys49Ala/Arg52Ala THB1 variants were prepared, and the consequences of the replacements on the Lys (de)coordination equilibrium were characterized experimentally for comparison with computational prediction. The results reinforced the dynamic role of protein-propionate interactions and strongly suggested that cleavage of the Fe-Lys53 bond and ensuing conformational rearrangement is facilitated by protonation of the amino group inside the distal cavity., (Copyright © 2021 Elsevier Inc. All rights reserved.)

THB1 is a monomeric truncated hemoglobin (TrHb) found in the cytoplasm of the green alga Chlamydomonas reinhardtii. The canonical heme coordination scheme in hemoglobins is a proximal histidine ligand and an open distal site. In THB1, the latter site is occupied by Lys53, which is likely to facilitate Fe(II)/Fe(III) redox cycling but hinders dioxygen binding, two features inherent to the NO dioxygenase activity of the protein. TrHb surveys show that a lysine at a position aligning with Lys53 is an insufficient determinant of coordination, and in this study, we sought to identify factors controlling lysine affinity for the heme iron. We solved the "Lys-off" X-ray structure of THB1, represented by the cyanide adduct of the Fe(III) protein, and hypothesized that interactions that differ between the known "Lys-on" structure and the Lys-off structure participate in the control of Lys53 affinity for the heme iron. We applied an experimental approach (site-directed mutagenesis, heme modification, pH titrations in the Fe(III) and Fe(II) states) and a computational approach (MD simulations in the Fe(II) state) to assess the role of heme propionate-protein interactions, distal helix capping, and the composition of the distal pocket. All THB1 modifications resulted in a weakening of lysine affinity and affected the coupling between Lys53 proton binding and heme redox potential. The results supported the importance of specific heme peripheral interactions for the pH stability of iron coordination and the ability of the protein to undergo redox reactions., (Copyright © 2021. Published by Elsevier Inc.)

Cystathionine β-synthase (CBS) is an enzyme involved in sulfur metabolism that catalyzes the pyridoxal phosphate-dependent condensation of homocysteine with serine or cysteine to form cystathionine and water or hydrogen sulfide (H 2 S), respectively. CBS possesses a b -type heme coordinated by histidine and cysteine. Fe(III)-CBS is inert toward exogenous ligands, while Fe(II)-CBS is reactive. Both Fe(III)- and Fe(II)-CBS are sensitive to mercury compounds. In this study, we describe the kinetics of the reactions with mercuric chloride (HgCl 2 ) and p -chloromercuribenzoic acid. These reactions were multiphasic and resulted in five-coordinate CBS lacking thiolate ligation, with six-coordinate species as intermediates. Computational QM/MM studies supported the feasibility of formation of species in which the thiolate is proximal to both the iron ion and the mercury compound. The reactions of Fe(II)-CBS were faster than those of Fe(III)-CBS. The observed rate constants of the first phase increased hyperbolically with concentration of the mercury compounds, with limiting values of 0.3-0.4 s -1 for Fe(III)-CBS and 40 ± 4 s -1 for Fe(II)-CBS. The data were interpreted in terms of alternative models of conformational selection or induced fit. Exposure of Fe(III)-CBS to HgCl 2 led to heme release and activity loss. Our study reveals the complexity of the interactions between mercury compounds and CBS., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

The relationships between conformational dynamics, stability and protein function are not obvious. Frataxin (FXN) is an essential protein that forms part of a supercomplex dedicated to the iron-sulfur (Fe-S) cluster assembly within the mitochondrial matrix. In humans, the loss of FXN expression or a decrease in its functionality results in Friedreich's Ataxia, a cardio-neurodegenerative disease. Recently, the way in which FXN interacts with the rest of the subunits of the supercomplex was uncovered. This opens a window to explore relationships between structural dynamics and function. In this study, we prepared a set of FXN variants spanning a broad range of conformational stabilities. Variants S160I, S160M and A204R were more stable than the wild-type and showed similar biological activity. Additionally, we prepared SILCAR, a variant that combines S160I, L203C and A204R mutations. SILCAR was 2.4 kcal mol -1 more stable and equally active. Some of the variants were significantly more resistant to proteolysis than the wild-type FXN. SILCAR showed the highest resistance, suggesting a more rigid structure. It was corroborated by means of molecular dynamics simulations. Relaxation dispersion NMR experiments comparing SILCAR and wild-type variants suggested similar internal motions in the microsecond to millisecond timescale. Instead, variant S157I showed higher denaturation resistance but a significant lower function, similarly to that observed for the FRDA variant N146K. We concluded that the contribution of particular side chains to the conformational stability of FXN might be highly subordinated to their impact on both the protein function and the stability of the functional supercomplex., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In recent years, the mammalian mitochondrial protein complex for iron-sulfur cluster assembly has been the focus of important studies. This is partly because of its high degree of relevance in cell metabolism and because mutations of the involved proteins are the cause of several human diseases. Cysteine desulfurase NFS1 is the key enzyme of the complex. At present, it is well-known that the active form of NFS1 is stabilized by the small protein ISD11. In this work, the structure of the human mitochondrial ACP-ISD11 heterodimer was determined at 2.0 Å resolution. ACP-ISD11 forms a cooperative unit stabilized by several ionic interactions, hydrogen bonds, and apolar interactions. The 4'-phosphopantetheine-acyl chain, which is covalently bound to ACP, interacts with several residues of ISD11, modulating together with ACP the foldability of ISD11. Recombinant human ACP-ISD11 was able to interact with the NFS1 desulfurase, thus yielding an active enzyme, and the NFS1/ACP-ISD11 core complex was activated by frataxin and ISCU proteins. Internal motions of ACP-ISD11 were studied by molecular dynamics simulations, showing the persistence of the interactions between both protein chains. The conformation of the dimer is similar to that found in the context of the (NFS1/ACP-ISD11) 2 supercomplex core, which contains the Escherichia coli ACP instead of the human variant. This fact suggests a sequential mechanism for supercomplex consolidation, in which the ACP-ISD11 complex may fold independently and, after that, the NFS1 dimer would be stabilized.

Computer simulation studies of the molecular basis for ligand migration in proteins allow the description of key events such as the transition between docking sites, displacement of existing ligands and solvent molecules, and open/closure of specific "gates", among others. In heme proteins, ligand migration from the solvent to the active site preludes the binding to the heme iron and triggers different functions. In this work, molecular dynamics simulations, a Markov State Model of migration and empirical kinetic equations are combined to study the migration of O 2 and NO in two truncated hemoglobins of Mycobacterium tuberculosis (Mt-TrHbN and Mt-TrHbO). For Mt-TrHbN, we show that the difference in the association constant in the oxy and deoxy states relies mainly in the displacement of water molecules anchored in the distal cavity in the deoxy form. The results here provide a valuable approach to study ligand migration in globins., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Toxic chemicals within and adsorbed to microplastics (0.05-5 mm) have the potential to biomagnify in food webs. However, microplastic concentrations in highly productive, coastal habitats are not well understood. Therefore, we quantified the presence of microplastics in a benthic community and surrounding environment of a remote marine reserve on the open coast of California, USA. Concentrations of microplastic particles in seawater were 36.59 plastics/L and in sediments were 0.227 ± 0.135 plastics/g. Densities of microplastics on the surfaces of two morphologically distinct species of macroalgae were 2.34 ± 2.19 plastics/g (Pelvetiopsis limitata) and 8.65 ± 6.44 plastics/g (Endocladia muricata). Densities were highest in the herbivorous snail, Tegula funebralis, at 9.91 ± 6.31 plastics/g, potentially due to bioaccumulation. This study highlights the need for further investigations of the prevalence and potential harm of microplastics in benthic communities at remote locations as well as human population centers., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Proteins are sensitive to temperature, and abrupt changes in the normal temperature conditions can have a profound impact on both structure and function, leading to protein unfolding. However, the adaptation of certain organisms to extreme conditions raises questions about the structural features that permit the structure and function of proteins to be preserved under these adverse conditions. To gain insight into the molecular basis of protein thermostability in the globin family, we have examined three representative examples: human neuroglobin, horse heart myoglobin, and Drosophila hemoglobin, which differ in their melting temperatures and coordination states of the heme iron in the absence of external ligands. In order to elucidate the possible mechanisms that govern the thermostability of these proteins, microsecond-scale classical molecular dynamics simulations were performed at different temperatures. Structural fluctuations and essential dynamics were analyzed, indicating that the flexibility of the CD region, which includes the two short C and D helixes and the connecting CD loop, is directly related to the thermostability. We observed that a larger inherent flexibility of the protein produces higher thermostability, probably concentrating the thermal fluctuations observed at high temperature in flexible regions, preventing unfolding. Globally, the results of this work improve our understanding of thermostability in the globin family.

Extensins (EXTs) are highly repetitive plant O-glycoproteins that require several post-translational modifications (PTMs) to become functional in plant cell walls. First, they are hydroxylated on contiguous proline residues; then they are O-glycosylated on hydroxyproline and serine. After secretion into the apoplast, O-glycosylated EXTs form a tridimensional network organized by inter- and intra-Tyr linkages. Recent studies have made significant progress in the identification of the enzymatic machinery required to process EXTs, which includes prolyl 4-hydroxylases, glycosyltransferases, papain-type cysteine endopeptidases, and peroxidases. EXTs are abundant in plant tissues and are particularly important in rapidly expanding root hairs and pollen tubes, which grow in a polar manner. Small changes in EXT PTMs affect fast-growing cells, although the molecular mechanisms underlying this regulation are unknown. In this review, we highlight recent advances in our understanding of EXT modifications throughout the secretory pathway, EXT assembly in cell walls, and possible sensing mechanisms involving the Catharanthus roseus cell surface sensor receptor-like kinases located at the interface between the apoplast and the cytoplasmic side of the plasma membrane., (Copyright © 2018 The Author. Published by Elsevier Inc. All rights reserved.)

Human hemoglobin (Hb) is a benchmark protein of structural biology that shaped our view of allosterism over 60 years ago, with the introduction of the MWC model based on Perutz structures of the oxy(R) and deoxy(T) states and the more recent Tertiary Two-State model that proposed the existence of individual subunit states -"r" and "t"-, whose structure is yet unknown. Cooperative oxygen binding is essential for Hb function, and despite decades of research there are still open questions related to how tertiary and quaternary changes regulate oxygen affinity. In the present work, we have determined the free energy profiles of oxygen migration and for HisE7 gate opening, with QM/MM calculations of the oxygen binding energy in order to address the influence of tertiary differences in the control of oxygen affinity. Our results show that in the α subunit the low to high affinity transition is achieved by a proximal effect that mostly affects oxygen dissociation and is the driving force of the allosteric transition, while in the β subunit the affinity change results from a complex interplay of proximal and distal effects, including an increase in the HE7 gate opening, that as shown by free energy profiles promotes oxygen uptake.

Heme proteins are ubiquitous in nature and perform many diverse functions in all kingdoms of life. Many of these functions are related to large-scale conformational transitions and allosteric processes. Sampling of these large conformational changes is computationally very challenging. In this context, coarse-grain simulations emerge as an efficient approach to explore the conformational landscape. In this work, we present a coarse-grained model of the heme group and thoroughly validate this model in different benchmark examples, which include the monomeric heme proteins myoglobin and neuroglobin and the tetrameric human hemoglobin where we evaluated the method's ability to explore conformational changes (as the formation of hexacoordinated species) and allosteric transitions (as the well-known R → T transition). The obtained results are compared with atomistic molecular dynamics simulations. Overall, the results indicate that this approach conserves the essential dynamical information on different allosteric processes.

Human indoleamine 2,3-dioxygenase catalyzes the oxidative cleavage of tryptophan to N-formyl kynurenine, the initial and rate-limiting step in the kynurenine pathway. Additionally, this enzyme has been identified as a possible target for cancer therapy. A 20-amino acid protein segment (the JK loop), which connects the J and K helices, was not resolved in the reported hIDO crystal structure. Previous studies have shown that this loop undergoes structural rearrangement upon substrate binding. In this work, we apply a combination of replica exchange molecular dynamics simulations and site-directed mutagenesis experiments to characterize the structure and dynamics of this protein region. Our simulations show that the JK loop can be divided into two regions: the first region (JK loop(C)) displays specific and well-defined conformations and is within hydrogen bonding distance of the substrate, while the second region (JK loop(N)) is highly disordered and exposed to the solvent. The peculiar flexible nature of JK loop(N) suggests that it may function as a target for post-translational modifications and/or a mediator for protein-protein interactions. In contrast, hydrogen bonding interactions are observed between the substrate and Thr379 in the highly conserved "GTGG" motif of JK loop(C), thereby anchoring JK loop(C) in a closed conformation, which secures the appropriate substrate binding mode for catalysis. Site-directed mutagenesis experiments confirm the key role of this residue, highlighting the importance of the JK loop(C) conformation in regulating the enzymatic activity. Furthermore, the existence of the partially and totally open conformations in the substrate-free form suggests a role of JK loop(C) in controlling substrate and product dynamics.

Unlabelled: Coarse grain (CG) models allow long-scale simulations with a much lower computational cost than that of all-atom simulations. However, the absence of atomistic detail impedes the analysis of specific atomic interactions that are determinant in most interesting biomolecular processes. In order to study these phenomena, it is necessary to reconstruct the atomistic structure from the CG representation. This structure can be analyzed by itself or be used as an onset for atomistic molecular dynamics simulations. In this work, we present a computer program that accurately reconstructs the atomistic structure from a CG model for proteins, using a simple geometrical algorithm., Availability and Implementation: The software is free and available online at http://www.ic.fcen.uba.ar/cg2aa/cg2aa.py, Supplementary Information: Supplementary data are available at Bioinformatics online., Contact: lula@qi.fcen.uba.ar., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Myoglobin (Mb) and neuroglobin (Ngb) are representative members of pentacoordinated and bis-histidyl, hexacoordinated globins. In spite of their low sequence identity, they show surprisingly similar three-dimensional folds. The ability of Ngb to form a hexacoordinated bis-histidyl complex with the distal HisE7 has a strong impact on ligand affinity. The factors governing such different behaviors have not been completely understood yet, even though they are extremely relevant to establish structure-function relationships within the globin superfamily., Methods: In this work we generated chimeric proteins by swapping a previously identified regulatory segment - the CD region - and evaluated comparatively the structural and functional properties of the resulting proteins by molecular dynamics simulations, and spectroscopic and kinetic investigations., Results: Our results show that chimeric proteins display heme coordination properties displaced towards those expected for the corresponding CD region. In particular, in the absence of exogenous ligands, chimeric Mb is found as a partially hexacoordinated bis-histidyl species, whereas chimeric Ngb shows a lower equilibrium constant for forming a hexacoordinated bis-histidyl species., Conclusions: While these results confirm the regulatory role of the CD region for bis-histidyl hexacoordination, they also suggest that additional sources contribute to fine tune the equilibrium. General significance Globins constitute a ubiquitous group of heme proteins widely found in all kingdoms of life. These findings raise challenging questions regarding the structure-function relationships in these proteins, as bis-histidyl hexacoordination emerges as a novel regulatory mechanism of the physiological function of globins., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The HIV-1 Tat protein and several small molecules bind to HIV-1 transactivation responsive RNA (TAR) by selecting sparsely populated but pre-existing conformations. Thus, a complete characterization of TAR conformational ensemble and dynamics is crucial to understand this paradigmatic system and could facilitate the discovery of new antivirals targeting this essential regulatory element. We show here that molecular dynamics simulations can be effectively used toward this goal by bridging the gap between functionally relevant time scales that are inaccessible to current experimental techniques. Specifically, we have performed several independent microsecond long molecular simulations of TAR based on one of the most advanced force fields available for RNA, the parmbsc0 AMBER. Our simulations are first validated against available experimental data, yielding an excellent agreement with measured residual dipolar couplings and order parameter S(2). This contrast with previous molecular dynamics simulations (Salmon et al., J. Am. Chem. Soc. 2013 135, 5457-5466) based on the CHARMM36 force field, which could achieve only modest accord with the experimental RDC values. Next, we direct the computation toward characterizing the internal dynamics of TAR over the microsecond time scale. We show that the conformational fluctuations observed over this previously elusive time scale have a strong functionally oriented character in that they are primed to sustain and assist ligand binding.

In this work we review the application of classical and quantum-mechanical atomistic computer simulation tools to the investigation of small ligand interaction with globins. In the first part, studies of ligand migration, with its connection to kinetic association rate constants (kon), are presented. In the second part, we review studies for a variety of ligands such as O2, NO, CO, HS(-), F(-), and NO2(-) showing how the heme structure, proximal effects, and the interactions with the distal amino acids can modulate protein ligand binding. The review presents mainly results derived from our previous works on the subject, in the context of other theoretical and experimental studies performed by others. The variety and extent of the presented data yield a clear example of how computer simulation tools have, in the last decade, contributed to our deeper understanding of small ligand interactions with globins. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Bitter molecules in humans are detected by ∼25 G protein-coupled receptors (GPCRs). The lack of atomic resolution structure for any of them is complicating an in depth understanding of the molecular mechanisms underlying bitter taste perception. Here, we investigate the molecular determinants of the interaction of the TAS2R38 bitter taste receptor with its agonists phenylthiocarbamide (PTC) and propylthiouracil (PROP). We use the recently developed hybrid Molecular Mechanics/Coarse Grained (MM/CG) method tailored specifically for GPCRs. The method, through an extensive exploration of the conformational space in the binding pocket, allows the identification of several residues important for agonist binding that would have been very difficult to capture from the standard bioinformatics/docking approach. Our calculations suggest that both agonists bind to Asn103, Phe197, Phe264 and Trp201, whilst they do not interact with the so-called extra cellular loop 2, involved in cis-retinal binding in the GPCR rhodopsin. These predictions are consistent with data sets based on more than 20 site-directed mutagenesis and functional calcium imaging experiments of TAS2R38. The method could be readily used for other GPCRs for which experimental information is currently lacking.

Nonsymbiotic hemoglobins (nsHbs) form a widely distributed class of plant proteins, which function remains unknown. Despite the fact that class 1 plant nonsymbiotic hemoglobins are hexacoordinate (6c) heme proteins (hxHbs), their hexacoordination equilibrium constants are much lower than in hxHbs from animals or bacteria. In addition, they are characterized by having very high oxygen affinities and low oxygen dissociation rate constants. Rice hemoglobin 1 (rHb1) is a class 1 nonsymbiotic hemoglobin. It crystallizes as a fully associated homodimer with both subunits in 6c state, but showing slightly different conformations, thus leading to an asymmetric crystallographic homodimer. The residues that constitute the dimeric interface are conserved among all nsHbs, suggesting that the quaternary structure could be relevant to explain the chemical behavior and biological function of this family of proteins. In this work, we analyze the molecular basis that determine the hexacoordination equilibrium in rHb1. Our results indicate that dynamical features of the quaternary structure significantly affect the hexacoordination process. Specifically, we observe that the pentacoordinate state is stabilized in the dimer with respect to the isolated monomers. Moreover, the dimer behaves asymmetrically, in a negative cooperative scheme. The results presented in this work are fully consistent with our previous hypothesis about the key role played by the nature of the CD region in determining the coordination state of globins., (Copyright © 2013 Wiley Periodicals, Inc.)

Indoleamine 2,3-dioxygenase (IDO) and tryptophan dioxygenase (TDO) are two heme proteins that catalyze the oxidation reaction of tryptophan (Trp) to N-formylkynurenine (NFK). Human IDO (hIDO) has recently been recognized as a potent anticancer drug target, a fact that triggered intense research on the reaction and inhibition mechanisms of hIDO. Our recent studies revealed that the dioxygenase reaction catalyzed by hIDO and TDO is initiated by addition of the ferric iron-bound superoxide to the C(2)═C(3) bond of Trp to form a ferryl and Trp-epoxide intermediate, via a 2-indolenylperoxo radical transition state. The data demonstrate that the two atoms of dioxygen are inserted into the substrate in a stepwise fashion, challenging the paradigm of heme-based dioxygenase chemistry. In the current study, we used QM/MM methods to decipher the mechanism by which the second ferryl oxygen is inserted into the Trp-epoxide to form the NFK product in hIDO. Our results show that the most energetically favored pathway involves proton transfer from Trp-NH(3)(+) to the epoxide oxygen, triggering epoxide ring opening and a concerted nucleophilic attack of the ferryl oxygen to the C(2) of Trp that leads to a metastable reaction intermediate. This intermediate subsequently converts to NFK, following C(2)-C(3) bond cleavage and the associated back proton transfer from the oxygen to the amino group of Trp. A comparative study with Xantomonas campestris TDO (xcTDO) indicates that the reaction follows a similar pathway, although subtle differences distinguishing the two enzyme reactions are evident. The results underscore the importance of the NH(3)(+) group of Trp in the two-step ferryl-based mechanism of hIDO and xcTDO, by acting as an acid catalyst to facilitate the epoxide ring-opening reaction and ferryl oxygen addition to the indole ring.

Understanding how ligands bind to G-protein coupled receptors (GPCRs) provides insights into a myriad of cell processes and is crucial for drug development. Here we extend a hybrid molecular mechanics/coarse-grained (MM/CG) approach applied previously to enzymes to GPCR/ligand complexes. The accuracy of this method for structural predictions is established by comparison with recent atomistic molecular dynamics simulations on the human β2 adrenergic receptor, a member of the GPCRs superfamily. The results obtained with the MM/CG methodology show a good agreement with previous all-atom classical dynamics simulations, in particular in the structural description of the ligand binding site. This approach could be used for high-throughput predictions of ligand poses in a variety of GPCRs.

Tryptophan dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) are the only two heme proteins that catalyze the oxidation reaction of tryptophan (Trp) to N-formylkynurenine. While human IDO is able to oxidize both L- and D-Trp, human TDO (hTDO) displays major specificity for L-Trp. In this work, we aim to interrogate the molecular basis for the substrate stereoselectivity of hTDO. Our previous molecular dynamics simulation studies of Xanthomonas campestris TDO (xcTDO) showed that a hydrogen bond between T254 (T342 in hTDO) and the ammonium group of the substrate is present in the L-Trp-bound enzyme, but not in the D-Trp-bound enzyme. The fact that this is the only notable structural alteration induced by the change in the stereo structure of the substrate prompted us to produce and characterize the T342A mutant of hTDO to evaluate the structural role of T342 in controlling the substrate stereoselectivity of the enzyme. The experimental results indicate that the mutation only slightly perturbs the global structural properties of the enzyme but totally abolishes the substrate stereoselectivity. Molecular dynamics simulations of xcTDO show that T254 controls the substrate stereoselectivity of the enzyme by (i) modulating the hydrogen bonding interaction between the NH(3)(+) group and epoxide oxygen of the ferryl-indole 2,3-epoxide intermediate of the enzyme and (ii) regulating the dynamics of two active site loops, loop(250-260) and loop(117-130), critical for substrate binding.

Since proteins are dynamic systems in living organisms, the employment of methodologies contemplating this crucial characteristic results fundamental to allow revealing several aspects of their function. In this work, we present results obtained using classical mechanical atomistic simulation tools applied to understand the connection between protein dynamics and ligand migration. Firstly, we will present a review of the different sampling schemes used in the last years to obtain both ligand migration pathways and the thermodynamic information associated with the process. Secondly, we will focus on representative examples in which the schemes previously presented are employed, concerning the following: i) ligand migration, tunnels, and cavities in myoglobin and neuroglobin; ii) ligand migration in truncated hemoglobin members; iii) NO escape and conformational changes in nitrophorins; iv) ligand selectivity in catalase and hydrogenase; and v) larger ligand migration: the P450 and haloalkane dehalogenase cases. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Alkyl hydroperoxide reductase E (AhpE), a novel subgroup of the peroxiredoxin family, comprises Mycobacterium tuberculosis AhpE (MtAhpE) and AhpE-like proteins present in many bacteria and archaea, for which functional characterization is scarce. We previously reported that MtAhpE reacted ~10(3) times faster with peroxynitrite than with hydrogen peroxide, but the molecular reasons for that remained unknown. Herein, we investigated the oxidizing substrate specificity and the oxidative inactivation of the enzyme. In most cases, both peroxidatic thiol oxidation and sulfenic acid overoxidation followed a trend in which those peroxides with the lower leaving-group pK(a) reacted faster than others. These data are in agreement with the accepted mechanisms of thiol oxidation and support that overoxidation occurs through sulfenate anion reaction with the protonated peroxide. However, MtAhpE oxidation and overoxidation by fatty acid-derived hydroperoxides (~10(8) and 10(5) M(-1) s(-1), respectively, at pH 7.4 and 25°C) were much faster than expected according to the Brønsted relationship with leaving-group pK(a). A stoichiometric reduction of the arachidonic acid hydroperoxide 15-HpETE to its corresponding alcohol was confirmed. Interactions of fatty acid hydroperoxides with a hydrophobic groove present on the reduced MtAhpE surface could be the basis of their surprisingly fast reactivity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The large apolar tunnel traversing the mini-hemoglobin from Cerebratulus lacteus (CerHb) has been examined by x-ray crystallography, ligand binding kinetics, and molecular dynamic simulations. The addition of 10 atm of xenon causes loss of diffraction in wild-type (wt) CerHbO(2) crystals, but Leu-86(G12)Ala CerHbO(2), which has an increased tunnel volume, stably accommodates two discrete xenon atoms: one adjacent to Leu-86(G12) and another near Ala-55(E18). Molecular dynamics simulations of ligand migration in wt CerHb show a low energy pathway through the apolar tunnel when Leu or Ala, but not Phe or Trp, is present at the 86(G12) position. The addition of 10-15 atm of xenon to solutions of wt CerHbCO and L86A CerHbCO causes 2-3-fold increases in the fraction of geminate ligand recombination, indicating that the bound xenon blocks CO escape. This idea was confirmed by L86F and L86W mutations, which cause even larger increases in the fraction of geminate CO rebinding, 2-5-fold decreases in the bimolecular rate constants for ligand entry, and large increases in the computed energy barriers for ligand movement through the apolar tunnel. Both the addition of xenon to the L86A mutant and oxidation of wt CerHb heme iron cause the appearance of an out Gln-44(E7) conformer, in which the amide side chain points out toward the solvent and appears to lower the barrier for ligand escape through the E7 gate. However, the observed kinetics suggest little entry and escape (≤ 25%) through the E7 pathway, presumably because the in Gln-44(E7) conformer is thermodynamically favored.

The first and rate-limiting step of the kynurenine pathway, in which tryptophan (Trp) is converted to N-formylkynurenine is catalyzed by two heme-containing proteins, Indoleamine 2,3-dioxygenase (IDO), and Tryptophan 2,3-dioxygenase (TDO). In mammals, TDO is found exclusively in liver tissue, IDO is found ubiquitously in all tissues. IDO has become increasingly popular in pharmaceutical research as it was found to be involved in many physiological situations, including immune escape of cancer. More importantly, small-molecule inhibitors of IDO are currently utilized in cancer therapy. One of the main concerns for the design of human IDO (hIDO) inhibitors is that they should be selective enough to avoid inhibition of TDO. In this work, we have used a combination of classical molecular dynamics (MD) and hybrid quantum-classical (QM/MM) methodologies to establish the structural basis that determine the differences in (a) the interactions of TDO and IDO with small ligands (CO/O(2)) and (b) the substrate stereo-specificity in hIDO and TDO. Our results indicate that the differences in small ligand bound structures of IDO and TDO arise from slight differences in the structure of the bound substrate complex. The results also show that substrate stereo-specificity of TDO is achieved by the perfect fit of L-Trp, but not D-Trp, which exhibits weaker interactions with the protein matrix. For hIDO, the presence of multiple stable binding conformations for L/D-Trp reveal the existence of a large and dynamic active site. Taken together, our data allow determination of key interactions useful for the future design of more potent hIDO-selective inhibitors., (© 2010 Wiley-Liss, Inc.)

Tryptophan dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) are two heme-containing enzymes which catalyze the conversion of L: -tryptophan to N-formylkynurenine (NFK). In mammals, TDO is mostly expressed in liver and is involved in controlling homeostatic serum tryptophan concentrations, whereas IDO is ubiquitous and is involved in modulating immune responses. Previous studies suggested that the first step of the dioxygenase reaction involves the deprotonation of the indoleamine group of the substrate by an evolutionarily conserved distal histidine residue in TDO and the heme-bound dioxygen in IDO. Here, we used classical molecular dynamics and hybrid quantum mechanical/molecular mechanical methods to evaluate the base-catalyzed mechanism. Our data suggest that the deprotonation of the indoleamine group of the substrate by either histidine in TDO or heme-bound dioxygen in IDO is not energetically favorable. Instead, the dioxygenase reaction can be initiated by a direct attack of heme-bound dioxygen on the C(2)=C(3) bond of the indole ring, leading to a protein-stabilized 2,3-alkylperoxide transition state and a ferryl epoxide intermediate, which subsequently recombine to generate NFK. The novel sequential two-step oxygen addition mechanism is fully supported by our recent resonance Raman data that allowed identification of the ferryl intermediate (Lewis-Ballester et al. in Proc Natl Acad Sci USA 106:17371-17376, 2009). The results reveal the subtle differences between the TDO and IDO reactions and highlight the importance of protein matrix in modulating stereoelectronic factors for oxygen activation and the stabilization of both transition and intermediate states.

In contrast to the wide spectrum of cytochrome P450 monooxygenases, there are only 2 heme-based dioxygenases in humans: tryptophan dioxygenase (hTDO) and indoleamine 2,3-dioxygenase (hIDO). hTDO and hIDO catalyze the same oxidative ring cleavage reaction of L-tryptophan to N-formyl kynurenine, the initial and rate-limiting step of the kynurenine pathway. Despite immense interest, the mechanism by which the 2 enzymes execute the dioxygenase reaction remains elusive. Here, we report experimental evidence for a key ferryl intermediate of hIDO that supports a mechanism in which the 2 atoms of dioxygen are inserted into the substrate via a consecutive 2-step reaction. This finding introduces a paradigm shift in our understanding of the heme-based dioxygenase chemistry, which was previously believed to proceed via simultaneous incorporation of both atoms of dioxygen into the substrate. The ferryl intermediate is not observable during the hTDO reaction, highlighting the structural differences between the 2 dioxygenases, as well as the importance of stereoelectronic factors in modulating the reactions.

Since the discovery of soluble guanylate cyclase (sGC) as the mammalian receptor for nitric oxide (NO), numerous studies have been performed in order to understand how sGC transduces the NO signal. However, the structural basis of sGC activation is still not completely elucidated. Spectroscopic and kinetic studies showed that the key step in the activation mechanism was the NO-induced breaking of the iron proximal histidine bond in the so-called 6c-NO to 5c-NO transition. The main breakthrough in the understanding of sGC activation mechanism came, however, from the elucidation of crystal structures for two different prokaryotic heme NO oxygen (HNOX) domains, which are homologues to the sGC heme domain. In this work we present computer simulation results of Thermoanaerobacter tencogensis HNOX that complement these structural studies, yielding molecular explanations to several poorly understood properties of these proteins. Specifically, our results explain the differential ligand binding patterns of the HNOX domains according to the nature of proximal and distal residues. We also show that the natural dynamics of these proteins is intimately related with the proposed conformational dependent activation process, which involves mainly the alphaFbeta1 loop and the alphaA-alphaC distal subdomain. The results from the sGC models also support this view and suggest a key role for the alphaFbeta1 loop in the iron proximal histidine bond breaking process and, therefore, in the sGC activation mechanism.

This chapter reviews the application of classical and quantum-mechanical atomistic simulation tools used in the investigation of several relevant issues in nitric oxide reactivity with globins and presents different simulation strategies based on classical force fields: standard molecular dynamics, essential dynamics, umbrella sampling, multiple steering molecular dynamics, and a novel technique for exploring the protein energy landscape. It also presents hybrid quantum-classical schemes as a tool to obtain relevant information regarding binding energies and chemical reactivity of globins. As illustrative examples, investigations of the structural flexibility, ligand migration profiles, oxygen affinity, and reactivity toward nitric oxide of truncated hemoglobin N of Mycobacterium tuberculosis are presented.

The binding of diatomic ligands, such as O(2), NO, and CO, to heme proteins is a process intimately related with their function. In this work, we analyzed by means of a combination of classical Molecular Dynamics (MD) and Hybrid Quantum-Classical (QM/MM) techniques the existence of multiple conformations in the distal site of heme proteins and their influence on oxygen affinity regulation. We considered two representative examples: soybean leghemoglobin (Lba) and Paramecium caudatum truncated hemoglobin (PcHb). The results presented in this work provide a molecular interpretation for the kinetic, structural, and mutational data that cannot be obtained by assuming a single distal conformation., ((c) 2007 Wiley-Liss, Inc.)

Heme proteins are found in all living organisms, and perform a wide variety of tasks ranging from electron transport, to the oxidation of organic compounds, to the sensing and transport of small molecules. In this work we review the application of classical and quantum-mechanical atomistic simulation tools to the investigation of several relevant issues in heme proteins chemistry: (i) conformational analysis, ligand migration, and solvation effects studied using classical molecular dynamics simulations; (ii) electronic structure and spin state energetics of the active sites explored using quantum-mechanics (QM) methods; (iii) the interaction of heme proteins with small ligands studied through hybrid quantum mechanics-molecular mechanics (QM-MM) techniques; (iv) and finally chemical reactivity and catalysis tackled by a combination of quantum and classical tools.

Heme proteins are found in all living organisms and are capable of performing a wide variety of tasks, requiring in many cases the binding of diatomic ligands, namely, O(2), CO, and/or NO. Therefore, subtle regulation of these diatomic ligands' affinity is one of the key issues for determining a heme protein's function. This regulation is achieved through direct H-bond interactions between the bound ligand and the protein, and by subtle tuning of the intrinsic heme group reactivity. In this work, we present an investigation of the proximal regulation of oxygen affinity in Fe(II) histidine coordinated heme proteins by means of computer simulation. Density functional theory calculations on heme model systems are used to analyze three proximal effects: charge donation, rotational position, and distance to the heme porphyrin plane of the proximal histidine. In addition, hybrid quantum-classical (QM-MM) calculations were performed in two representative proteins: myoglobin and leghemoglobin. Our results show that all three effects are capable of tuning the Fe-O(2) bond strength in a cooperative way, consistently with the experimental data on oxygen affinity. The proximal effects described herein could operate in a large variety of O(2)-binding heme proteins-in combination with distal effects-and are essential to understand the factors determining a heme protein's O(2) affinity.