ABSTRACT
DNA gyrases catalyze negative supercoiling of DNA, are essential for bacterial DNA replication, transcription, and recombination, and are important antibacterial targets in multiple pathogens, including Mycobacterium tuberculosis, which in 2021 caused >1.5 million deaths worldwide. DNA gyrase is a tetrameric (A2B2) protein formed from two subunit types: gyrase A (GyrA) carries the breakage-reunion active site, whereas gyrase B (GyrB) catalyzes ATP hydrolysis required for energy transduction and DNA translocation. The GyrB ATPase domains dimerize in the presence of ATP to trap the translocated DNA (T-DNA) segment as a first step in strand passage, for which hydrolysis of one of the two ATPs and release of the resulting inorganic phosphate is rate-limiting. Here, dynamical-nonequilibrium molecular dynamics (D-NEMD) simulations of the dimeric 43 kDa N-terminal fragment of M. tuberculosis GyrB show how events at the ATPase site (dissociation/hydrolysis of bound nucleotides) are propagated through communication pathways to other functionally important regions of the GyrB ATPase domain. Specifically, our simulations identify two distinct pathways that respectively connect the GyrB ATPase site to the corynebacteria-specific C-loop, thought to interact with GyrA prior to DNA capture, and to the C-terminus of the GyrB transduction domain, which in turn contacts the C-terminal GyrB topoisomerase-primase (TOPRIM) domain responsible for interactions with GyrA and the centrally bound G-segment DNA. The connection between the ATPase site and the C-loop of dimeric GyrB is consistent with the unusual properties of M. tuberculosis DNA gyrase relative to those from other bacterial species.
Subject(s)
Adenosine Triphosphatases , DNA Gyrase , Molecular Dynamics Simulation , Mycobacterium tuberculosis , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/genetics , DNA Gyrase/metabolism , DNA Gyrase/chemistry , DNA Gyrase/genetics , Adenosine Triphosphatases/metabolism , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Protein Domains , Adenosine Triphosphate/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Signal TransductionABSTRACT
Molecular chaperones, a family of proteins of which Hsp90 and Hsp70 are integral members, form an essential machinery to maintain healthy proteomes by controlling the folding and activation of a plethora of substrate client proteins. This is achieved through cycles in which Hsp90 and Hsp70, regulated by task-specific co-chaperones, process ATP and become part of a complex network that undergoes extensive compositional and conformational variations. Despite impressive advances in structural knowledge, the mechanisms that regulate the dynamics of functional assemblies, their response to nucleotides, and their relevance for client remodeling are still elusive. Here, we focus on the glucocorticoid receptor (GR):Hsp90:Hsp70:co-chaperone Hop client-loading and the GR:Hsp90:co-chaperone p23 client-maturation complexes, key assemblies in the folding cycle of glucocorticoid receptor (GR), a client strictly dependent upon Hsp90/Hsp70 for activity. Using a combination of molecular dynamics simulation approaches, we unveil with unprecedented detail the mechanisms that underpin function in these chaperone machineries. Specifically, we dissect the processes by which the nucleotide-encoded message is relayed to the client and how the distinct partners of the assemblies cooperate to (pre)organize partially folded GR during Loading and Maturation. We show how different ligand states determine distinct dynamic profiles for the functional interfaces defining the interactions in the complexes and modulate their overall flexibility to facilitate progress along the chaperone cycle. Finally, we also show that the GR regions engaged by the chaperone machinery display peculiar energetic signatures in the folded state, which enhance the probability of partial unfolding fluctuations. From these results, we propose a model where a dynamic cross-talk emerges between the chaperone dynamics states and remodeling of client-interacting regions. This factor, coupled to the highly dynamic nature of the assemblies and the conformational heterogeneity of their interactions, provides the basis for regulating the functions of distinct assemblies during the chaperoning cycle.
ABSTRACT
ß-thalassemia is a prevalent genetic disorder causing severe anemia due to defective erythropoiesis, with few treatment options. Studying the underlying molecular defects is impeded by paucity of suitable patient material. In this study we create human disease cellular model systems for ß-thalassemia by gene editing the erythroid line BEL-A, which accurately recapitulate the phenotype of patient erythroid cells. We also develop a high throughput compatible fluorometric-based assay for evaluating severity of disease phenotype and utilize the assay to demonstrate that the lines respond appropriately to verified reagents. We next use the lines to perform extensive analysis of the altered molecular mechanisms in ß-thalassemia erythroid cells, revealing upregulation of a wide range of biological pathways and processes along with potential novel targets for therapeutic investigation. Overall, the lines provide a sustainable supply of disease cells as research tools for identifying therapeutic targets and as screening platforms for new drugs and reagents.
Subject(s)
beta-Thalassemia , Humans , beta-Thalassemia/genetics , beta-Thalassemia/therapy , Erythropoiesis/genetics , Erythroid Cells , PhenotypeABSTRACT
The electron-conducting circuitry of life represents an as-yet untapped resource of exquisite, nanoscale biomolecular engineering. Here, we report the characterization and structure of a de novo diheme "maquette" protein, 4D2, which we subsequently use to create an expanded, modular platform for heme protein design. A well-folded monoheme variant was created by computational redesign, which was then utilized for the experimental validation of continuum electrostatic redox potential calculations. This demonstrates how fundamental biophysical properties can be predicted and fine-tuned. 4D2 was then extended into a tetraheme helical bundle, representing a 7 nm molecular wire. Despite a molecular weight of only 24 kDa, electron cryomicroscopy illustrated a remarkable level of detail, indicating the positioning of the secondary structure and the heme cofactors. This robust, expressible, highly thermostable and readily designable modular platform presents a valuable resource for redox protein design and the future construction of artificial electron-conducting circuitry.
Subject(s)
Hemeproteins , Biophysics , Cryoelectron Microscopy , Electrons , Oxidation-ReductionABSTRACT
The target for humoral immunity, SARS-CoV-2 spike glycoprotein, has become the focus of vaccine research and development. Previous work demonstrated that the N-terminal domain (NTD) of SARS-CoV-2 spike binds biliverdin-a product of heme catabolism-causing a strong allosteric effect on the activity of a subset of neutralizing antibodies. Herein, we show that the spike glycoprotein is also able to bind heme (KD = 0.5 ± 0.2 µM). Molecular modeling indicated that the heme group fits well within the same pocket on the SARS-CoV-2 spike NTD. Lined by aromatic and hydrophobic residues (W104, V126, I129, F192, F194, I203, and L226), the pocket provides a suitable environment to stabilize the hydrophobic heme. Mutagenesis of N121 has a substantive effect on heme binding (KD = 3000 ± 220 µM), confirming the pocket as a major heme binding location of the viral glycoprotein. Coupled oxidation experiments in the presence of ascorbate indicated that the SARS-CoV-2 glycoprotein can catalyze the slow conversion of heme to biliverdin. The heme trapping and oxidation activities of the spike may allow the virus to reduce levels of free heme during infection to facilitate evasion of the adaptive and innate immunity.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Antibodies, Viral , Biliverdine , Receptors, Virus/metabolism , Antibodies, NeutralizingABSTRACT
The SARS-CoV-2 main protease (Mpro) plays an essential role in the coronavirus lifecycle by catalyzing hydrolysis of the viral polyproteins at specific sites. Mpro is the target of drugs, such as nirmatrelvir, though resistant mutants have emerged that threaten drug efficacy. Despite its importance, questions remain on the mechanism of how Mpro binds its substrates. Here, we apply dynamical nonequilibrium molecular dynamics (D-NEMD) simulations to evaluate structural and dynamical responses of Mpro to the presence and absence of a substrate. The results highlight communication between the Mpro dimer subunits and identify networks, including some far from the active site, that link the active site with a known allosteric inhibition site, or which are associated with nirmatrelvir resistance. They imply that some mutations enable resistance by altering the allosteric behavior of Mpro. More generally, the results show the utility of the D-NEMD technique for identifying functionally relevant allosteric sites and networks including those relevant to resistance.
ABSTRACT
The SARS-CoV-2 spike protein contains a functionally important fatty acid (FA) binding site, which is also found in some other coronaviruses, e.g. SARS-CoV and MERS-CoV. The occupancy of the FA site by linoleic acid (LA) reduces infectivity by 'locking' the spike in a less infectious conformation. Here, we use dynamical-nonequilibrium molecular dynamics (D-NEMD) simulations to compare the allosteric responses of spike variants to LA removal. D-NEMD simulations show that the FA site is coupled to other functional regions of the protein, e.g. the receptor-binding motif (RBM), N-terminal domain (NTD), furin cleavage site, and regions surrounding the fusion peptide. D-NEMD simulations also identify the allosteric networks connecting the FA site to these functional regions. The comparison between the wild-type spike and four variants (Alpha, Delta, Delta plus, and Omicron BA.1) shows that the variants differ significantly in their responses to LA removal. The allosteric connections to the FA site on Alpha are generally similar to those on the wild-type protein, with the exception of the RBM and the S71-R78 region, which show a weaker link to the FA site. In contrast, Omicron is the most different variant, exhibiting significant differences in the RBM, NTD, V622-L629, and furin cleavage site. These differences in the allosteric modulation may be of functional relevance, potentially affecting transmissibility and virulence. Experimental comparison of the effects of LA on SARS-CoV-2 variants, including emerging variants, is warranted.
Subject(s)
COVID-19 , Humans , Furin/genetics , Linoleic Acid , SARS-CoV-2/geneticsABSTRACT
Targeting the colchicine binding site of α/ß tubulin microtubules can lead to suppression of microtubule dynamics, cell cycle arrest and apoptosis. Therefore, the development of microtubule (MT) inhibitors is considered a promising route to anticancer agents. Our approach to identify novel scaffolds as MT inhibitors depends on a 3D-structure-based pharmacophore approach and docking using three programs MOE, Autodock and BUDE (Bristol University Docking Engine) to screen a library of virtual compounds. From this work we identified the compound 7-(3-hydroxy-4-methoxy-phenyl)-3-(3-trifluoromethyl-phenyl)-6,7-dihydro-3H-imidazo[4,5-b]pyridin-5-ol (6) as a novel inhibitor scaffold. This compound inhibited several types of cancer cell proliferation at low micromolar concentrations with low toxicity. Compound 6 caused cell cycle arrest in the G2/M phase and blocked tubulin polymerization at low micromolar concentration (IC50 = 6.1 ±0.1 µM), inducing apoptosis via activation of caspase 9, increasing the level of the pro-apoptotic protein Bax and decreasing the level of the anti-apoptotic protein Bcl2. In summary, our approach identified a lead compound with potential antimitotic and antiproliferative activity.
ABSTRACT
Transport of proteins across and into membranes is a fundamental biological process with the vast majority being conducted by the ubiquitous Sec machinery. In bacteria, this is usually achieved when the SecY-complex engages the cytosolic ATPase SecA (secretion) or translating ribosomes (insertion). Great strides have been made towards understanding the mechanism of protein translocation. Yet, important questions remain - notably, the nature of the individual steps that constitute transport, and how the proton-motive force (PMF) across the plasma membrane contributes. Here, we apply a recently developed high-resolution protein transport assay to explore these questions. We find that pre-protein transport is limited primarily by the diffusion of arginine residues across the membrane, particularly in the context of bulky hydrophobic sequences. This specific effect of arginine, caused by its positive charge, is mitigated for lysine which can be deprotonated and transported across the membrane in its neutral form. These observations have interesting implications for the mechanism of protein secretion, suggesting a simple mechanism through which the PMF can aid transport by enabling a 'proton ratchet', wherein re-protonation of exiting lysine residues prevents channel re-entry, biasing transport in the outward direction.
Subject(s)
Escherichia coli Proteins , Arginine/metabolism , Bacterial Proteins/metabolism , Cell Membrane/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Lysine/metabolism , Protein Transport , SEC Translocation Channels/metabolismABSTRACT
As the global burden of SARS-CoV-2 infections escalates, so does the evolution of viral variants with increased transmissibility and pathology. In addition to this entrenched diversity, RNA viruses can also display genetic diversity within single infected hosts with co-existing viral variants evolving differently in distinct cell types. The BriSΔ variant, originally identified as a viral subpopulation from SARS-CoV-2 isolate hCoV-19/England/02/2020, comprises in the spike an eight amino-acid deletion encompassing a furin recognition motif and S1/S2 cleavage site. We elucidate the structure, function and molecular dynamics of this spike providing mechanistic insight into how the deletion correlates to viral cell tropism, ACE2 receptor binding and infectivity of this SARS-CoV-2 variant. Our results reveal long-range allosteric communication between functional domains that differ in the wild-type and the deletion variant and support a view of SARS-CoV-2 probing multiple evolutionary trajectories in distinct cell types within the same infected host.
Subject(s)
SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Animals , COVID-19/virology , Cell Line , Cryoelectron Microscopy , Evolution, Molecular , Furin/metabolism , Humans , Linoleic Acid/metabolism , Molecular Dynamics Simulation , Mutation , Protein Binding , Protein Conformation , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Viral Tropism , Virus InternalizationABSTRACT
ABSTRACT: A dynamical approach to nonequilibrium molecular dynamics (D-NEMD), proposed in the 1970s by Ciccotti et al., is undergoing a renaissance and is having increasing impact in the study of biological macromolecules. This D-NEMD approach, combining MD simulations in stationary (in particular, equilibrium) and nonequilibrium conditions, allows for the determination of the time-dependent structural response of a system using the Kubo-Onsager relation. Besides providing a detailed picture of the system's dynamic structural response to an external perturbation, this approach also has the advantage that the statistical significance of the response can be assessed. The D-NEMD approach has been used recently to identify a general mechanism of inter-domain signal propagation in nicotinic acetylcholine receptors, and allosteric effects in ß -lactamase enzymes, for example. It complements equilibrium MD and is a very promising approach to identifying and analysing allosteric effects. Here, we review the D-NEMD approach and its application to biomolecular systems, including transporters, receptors, and enzymes.
ABSTRACT
ATP binding cassette (ABC) transporters employ ATP hydrolysis to harness substrate translocation across membranes. The Escherichia coli MalFGK2E maltose importer is an example of a type I ABC importer and a model system for this class of ABC transporters. The MalFGK2E importer is responsible for the intake of malto-oligossacharides in E.coli. Despite being extensively studied, little is known about the effect of ATP hydrolysis and nucleotide exit on substrate transport. In this work, we studied this phenomenon using extensive molecular dynamics simulations (MD) along with potential of mean force calculations of maltose transport across the pore, in the pre-hydrolysis, post-hydrolysis and nucleotide-free states. We concluded that ATP hydrolysis and nucleotide exit trigger conformational changes that result in the decrease of energetic barriers to maltose translocation towards the cytoplasm, with a concomitant increase of the energy barrier in the periplasmic side of the pore, contributing for the irreversibility of the process. We also identified key residues that aid in positioning and orientation of maltose, as well as a novel binding pocket for maltose in MalG. Additionally, ATP hydrolysis leads to conformations similar to the nucleotide-free state. This study shows the contribution of ATP hydrolysis and nucleotide exit in the transport cycle, shedding light on ABC type I importer mechanisms.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Adenosine Triphosphate/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli , Hydrolysis , Maltose/metabolism , Protein ConformationABSTRACT
Changeux et al. (Changeux et al. C. R. Biol. 343:33-39.) recently suggested that the SARS-CoV-2 spike protein may interact with nicotinic acetylcholine receptors (nAChRs) and that such interactions may be involved in pathology and infectivity. This hypothesis is based on the fact that the SARS-CoV-2 spike protein contains a sequence motif similar to known nAChR antagonists. Here, we use molecular simulations of validated atomically detailed structures of nAChRs and of the spike to investigate the possible binding of the Y674-R685 region of the spike to nAChRs. We examine the binding of the Y674-R685 loop to three nAChRs, namely the human α4ß2 and α7 subtypes and the muscle-like αßγδ receptor from Tetronarce californica. Our results predict that Y674-R685 has affinity for nAChRs. The region of the spike responsible for binding contains a PRRA motif, a four-residue insertion not found in other SARS-like coronaviruses. The conformational behavior of the bound Y674-R685 is highly dependent on the receptor subtype; it adopts extended conformations in the α4ß2 and α7 complexes but is more compact when bound to the muscle-like receptor. In the α4ß2 and αßγδ complexes, the interaction of Y674-R685 with the receptors forces the loop C region to adopt an open conformation, similar to other known nAChR antagonists. In contrast, in the α7 complex, Y674-R685 penetrates deeply into the binding pocket in which it forms interactions with the residues lining the aromatic box, namely with TrpB, TyrC1, and TyrC2. Estimates of binding energy suggest that Y674-R685 forms stable complexes with all three nAChR subtypes. Analyses of simulations of the glycosylated spike show that the Y674-R685 region is accessible for binding. We suggest a potential binding orientation of the spike protein with nAChRs, in which they are in a nonparallel arrangement to one another.
Subject(s)
Receptors, Nicotinic/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Glycosylation , Humans , Molecular Dynamics Simulation , Peptides/chemistry , Peptides/metabolism , Protein Binding , Receptors, Nicotinic/chemistry , Spike Glycoprotein, Coronavirus/chemistry , ThermodynamicsABSTRACT
Changeux et al. recently suggested that the SARS-CoV-2 spike (S) protein may interact with nicotinic acetylcholine receptors (nAChRs). Such interactions may be involved in pathology and infectivity. Here, we use molecular simulations of validated atomically detailed structures of nAChRs, and of the S protein, to investigate this 'nicotinic hypothesis'. We examine the binding of the Y674-R685 loop of the S protein to three nAChRs, namely the human α4ß2 and α7 subtypes and the muscle-like αßγδ receptor from Tetronarce californica. Our results indicate that Y674-R685 has affinity for nAChRs and the region responsible for binding contains the PRRA motif, a four-residue insertion not found in other SARS-like coronaviruses. In particular, R682 has a key role in the stabilisation of the complexes as it forms interactions with loops A, B and C in the receptor's binding pocket. The conformational behaviour of the bound Y674-R685 region is highly dependent on the receptor subtype, adopting extended conformations in the α4ß2 and α7 complexes and more compact ones when bound to the muscle-like receptor. In the α4ß2 and αßγδ complexes, the interaction of Y674-R685 with the receptors forces the loop C region to adopt an open conformation similar to other known nAChR antagonists. In contrast, in the α7 complex, Y674-R685 penetrates deeply into the binding pocket where it forms interactions with the residues lining the aromatic box, namely with TrpB, TyrC1 and TyrC2. Estimates of binding energy suggest that Y674-R685 forms stable complexes with all three nAChR subtypes. Analyses of the simulations of the full-length S protein show that the Y674-R685 region is accessible for binding, and suggest a potential binding orientation of the S protein with nAChRs.
ABSTRACT
MCR (mobile colistin resistance) enzymes catalyse phosphoethanolamine (PEA) addition to bacterial lipid A, threatening the "last-resort" antibiotic colistin. Molecular dynamics and density functional theory simulations indicate that monozinc MCR supports PEA transfer to the Thr285 acceptor, positioning MCR as a mono- rather than multinuclear member of the alkaline phosphatase superfamily.
Subject(s)
Alkaline Phosphatase/chemistry , Anti-Bacterial Agents/chemistry , Bacterial Proteins/chemistry , Colistin/chemistry , Drug Resistance, Bacterial , Zinc/chemistry , Ethanolamines/chemistry , Lipid A/chemistry , Molecular Dynamics SimulationABSTRACT
Nicotinic acetylcholine receptors (nAChRs) modulate synaptic transmission in the nervous system. These receptors have emerged as therapeutic targets in drug discovery for treating several conditions, including Alzheimer's disease, pain, and nicotine addiction. In this in silico study, we use a combination of equilibrium and nonequilibrium molecular dynamics simulations to map dynamic and structural changes induced by nicotine in the human α4ß2 nAChR. They reveal a striking pattern of communication between the extracellular binding pockets and the transmembrane domains (TMDs) and show the sequence of conformational changes associated with the initial steps in this process. We propose a general mechanism for signal transduction for Cys-loop receptors: the mechanistic steps for communication proceed firstly through loop C in the principal subunit, and are subsequently transmitted, gradually and cumulatively, to loop F of the complementary subunit, and then to the TMDs through the M2-M3 linker.
Subject(s)
Lipid Bilayers/chemistry , Nicotine/chemistry , Phosphatidylcholines/chemistry , Protein Subunits/chemistry , Receptors, Nicotinic/chemistry , Signal Transduction , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Humans , Hydrogen-Ion Concentration , Ligands , Lipid Bilayers/metabolism , Molecular Dynamics Simulation , Nicotine/metabolism , Phosphatidylcholines/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Subunits/metabolism , Receptors, Nicotinic/metabolism , ThermodynamicsABSTRACT
Type-II NADH:quinone oxidoreductases (NDH-2s) are membrane proteins involved in respiratory chains and the only enzymes with NADH:quinone oxidoreductase activity expressed in Staphylococcus aureus (S. aureus), one of the most common causes of clinical infections. NDH-2s are members of the two-Dinucleotide Binding Domains Flavoprotein (tDBDF) superfamily, having a flavin adenine dinucleotide, FAD, as prosthetic group and NAD(P)H as substrate. The establishment of a Charge-Transfer Complex (CTC) between the isoalloxazine ring of the reduced flavin and the nicotinamide ring of NAD+ in NDH-2 was described, and in this work we explored its role in the kinetic mechanism using different electron donors and electron acceptors. We observed that CTC slows down the rate of the second half reaction (quinone reduction) and determines the effect of HQNO, an inhibitor. Also, protonation equilibrium simulations clearly indicate that the protonation probability of an important residue for proton transfer to the active site (D302) is influenced by the presence of the CTC. We propose that CTC is critical for the overall mechanism of NDH-2 and possibly relevant to keep a low quinol/quinone ratio and avoid excessive ROS production in vivo.
Subject(s)
Electron Transport , NAD(P)H Dehydrogenase (Quinone)/chemistry , Reactive Oxygen Species/metabolism , Staphylococcus aureus/enzymology , Binding Sites , Catalytic Domain , Flavin-Adenine Dinucleotide/chemistry , Flavin-Adenine Dinucleotide/metabolism , Kinetics , NAD(P)H Dehydrogenase (Quinone)/metabolism , Quinones/chemistry , Quinones/metabolism , Reactive Oxygen Species/chemistry , Staphylococcus aureus/pathogenicity , Substrate SpecificityABSTRACT
The ancient metabolism of photoferrotrophy is likely to have played a key role in the biogeochemical cycle of iron on Early Earth leading to the deposition of Banded Iron Formations prior to the emergence of oxygenic photosynthesis. Extant organisms still performing this metabolism provide a convenient window to peer into its molecular mechanisms. Here we report the molecular structure of FoxE, the putative terminal iron oxidase of Rhodobacter ferrooxidans SW2. This protein is organized as a trimer with two hemes and a disulfide bridge per monomer. The distance between hemes, their solvent exposure and the surface electrostatics ensure a controlled electron transfer rate. They also guarantee segregation between electron capture from ferrous iron and electron release to downstream acceptors, which do not favor the precipitation of ferric iron. Combined with the functional characterization of this protein, the structure reveals how iron oxidation can be performed in the periplasmic space of this Gram-negative bacterium at circumneutral pH, while minimizing the risk of mineral precipitation and cell encrustation.
Subject(s)
Ferrous Compounds/chemistry , Iron/chemistry , Oxidoreductases/chemistry , Rhodobacter/chemistry , Amino Acid Sequence , Disulfides/chemistry , Electron Transport/physiology , Electrons , Heme/chemistry , Molecular Structure , Oxidation-Reduction , Oxygen/chemistry , Photosynthesis/physiologyABSTRACT
Type II NADH:quinone oxidoreductases (NDH-2s) are membrane bound enzymes that deliver electrons to the respiratory chain by oxidation of NADH and reduction of quinones. In this way, these enzymes also contribute to the regeneration of NAD+, allowing several metabolic pathways to proceed. As for the other members of the two-Dinucleotide Binding Domains Flavoprotein (tDBDF) superfamily, the enzymatic mechanism of NDH-2s is still little explored and elusive. In this work we addressed the role of the conserved glutamate 172 (E172) residue in the enzymatic mechanism of NDH-2 from Staphylococcus aureus. We aimed to test our earlier hypothesis that E172 plays a key role in proton transfer to allow the protonation of the quinone. For this we performed a complete biochemical characterization of the enzyme's variants E172A, E172Q and E172S. Our steady state kinetic measurements show a clear decrease in the overall reaction rate, and our substrate interaction studies indicate the binding of the two substrates is also affected by these mutations. Interestingly our fast kinetic results show quinone reduction is more affected than NADH oxidation. We have also determined the X-ray crystal structure of the E172S mutant (2.55Ǻ) and compared it with the structure of the wild type (2.32Ǻ). Together these results support our hypothesis for E172 being of central importance in the catalytic mechanism of NDH-2, which may be extended to other members of the tDBDF superfamily.
Subject(s)
Bacterial Proteins/metabolism , Benzoquinones/metabolism , Glutamic Acid/metabolism , NADH Dehydrogenase/metabolism , NAD/metabolism , Quinone Reductases/metabolism , Staphylococcus aureus/metabolism , Oxidation-Reduction , Protein Binding/physiologyABSTRACT
Type II NADH:quinone oxidoreductases (NDH-2s) are membrane proteins involved in respiratory chains. These proteins contribute indirectly to the establishment of the transmembrane difference of electrochemical potential by catalyzing the reduction of quinone by oxidation of NAD(P)H. NDH-2s are widespread enzymes being present in the three domains of life. In this work, we explored the catalytic mechanism of NDH-2 by investigating the common elements of all NDH-2s, based on the rationale that conservation of such elements reflects their structural/functional importance. We observed conserved sequence motifs and structural elements among 1762 NDH-2s. We identified two proton pathways possibly involved in the protonation of the quinone. Our results led us to propose the first catalytic mechanism for NDH-2 family, in which a conserved glutamate residue, E172 (in NDH-2 from Staphylococcus aureus) plays a key role in proton transfer to the quinone pocket. This catalytic mechanism may also be extended to the other members of the two-Dinucleotide Binding Domains Flavoprotein (tDBDF) superfamily, such as sulfide:quinone oxidoreductases.