ABSTRACT
Mycobacterium tuberculosis is the single most important global infectious disease killer and a World Health Organization critical priority pathogen for development of new antimicrobials. M. tuberculosis DNA gyrase is a validated target for anti-TB agents, but those in current use target DNA breakage-reunion, rather than the ATPase activity of the GyrB subunit. Here, virtual screening, subsequently validated by whole-cell and enzyme inhibition assays, was applied to identify candidate compounds that inhibit M. tuberculosis GyrB ATPase activity from the Specs compound library. This approach yielded six compounds: four carbazole derivatives (1, 2, 3, and 8), the benzoindole derivative 11, and the indole derivative 14. Carbazole derivatives can be considered a new scaffold for M. tuberculosis DNA gyrase ATPase inhibitors. IC50 values of compounds 8, 11, and 14 (0.26, 0.56, and 0.08 µM, respectively) for inhibition of M. tuberculosis DNA gyrase ATPase activity are 5-fold, 2-fold, and 16-fold better than the known DNA gyrase ATPase inhibitor novobiocin. MIC values of these compounds against growth of M. tuberculosis H37Ra are 25.0, 3.1, and 6.2 µg/mL, respectively, superior to novobiocin (MIC > 100.0 µg/mL). Molecular dynamics simulations of models of docked GyrB:inhibitor complexes suggest that hydrogen bond interactions with GyrB Asp79 are crucial for high-affinity binding of compounds 8, 11, and 14 to M. tuberculosis GyrB for inhibition of ATPase activity. These data demonstrate that virtual screening can identify known and new scaffolds that inhibit both M. tuberculosis DNA gyrase ATPase activity in vitro and growth of M. tuberculosis bacteria.
Subject(s)
Antitubercular Agents , DNA Gyrase , Indoles , Mycobacterium tuberculosis , Topoisomerase II Inhibitors , Adenosine Triphosphatases/antagonists & inhibitors , Adenosine Triphosphatases/metabolism , Antitubercular Agents/pharmacology , Antitubercular Agents/chemistry , DNA Gyrase/metabolism , DNA Gyrase/chemistry , Drug Discovery , Drug Evaluation, Preclinical , Indoles/pharmacology , Indoles/chemistry , Ligands , Microbial Sensitivity Tests , Molecular Docking Simulation , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/drug effects , Topoisomerase II Inhibitors/pharmacology , Topoisomerase II Inhibitors/chemistryABSTRACT
Epistasis, the non-additive effect of mutations, can provide combinatorial improvements to enzyme activity that substantially exceed the gains from individual mutations. Yet the molecular mechanisms of epistasis remain elusive, undermining our ability to predict pathogen evolution and engineer biocatalysts. Here we reveal how directed evolution of a ß-lactamase yielded highly epistatic activity enhancements. Evolution selected four mutations that increase antibiotic resistance 40-fold, despite their marginal individual effects (≤2-fold). Synergistic improvements coincided with the introduction of super-stochiometric burst kinetics, indicating that epistasis is rooted in the enzyme's conformational dynamics. Our analysis reveals that epistasis stemmed from distinct effects of each mutation on the catalytic cycle. The initial mutation increased protein flexibility and accelerated substrate binding, which is rate-limiting in the wild-type enzyme. Subsequent mutations predominantly boosted the chemical steps by fine-tuning substrate interactions. Our work identifies an overlooked cause for epistasis: changing the rate-limiting step can result in substantial synergy that boosts enzyme activity.
ABSTRACT
Methyl carboxylate esters promote the formation of dimethyl ether (DME) from the dehydration of methanol in H-ZSM-5 zeolite. We employ a multilevel quantum method to explore the possible associative and dissociative mechanisms in the presence, and absence, of six methyl ester promoters. This hybrid method combines density functional theory, with dispersion corrections (DFT-D3), for the full periodic system, with second-order Møller-Plesset perturbation theory (MP2) for small clusters representing the reaction site, and coupled cluster with single, double, and perturbative triple substitution (CCSD(T)) for the reacting molecules. The calculated adsorption enthalpy of methanol, and reaction enthalpies of the dehydration of methanol to DME within H-ZSM-5, agree with experiment to within chemical accuracy (â¼4 kJ mol-1). For the promoters, a reaction pathway via an associative mechanism gives lower overall reaction enthalpies and barriers compared to the reaction with methanol only. Each stage of this mechanism is explored and related to experimental data. We provide evidence that suggests the promoter's adsorption to the Brønsted acid site is the most important factor dictating its efficiency.
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
Sire is a Python/C++ library that is used both to prototype new algorithms and as an interoperability engine for exchanging information between molecular simulation programs. It provides a collection of file parsers and information converters that together make it easier to combine and leverage the functionality of many other programs and libraries. This empowers researchers to use sire to write a single script that can, for example, load a molecule from a PDBx/mmCIF file via Gemmi, perform SMARTS searches via RDKit, parameterize molecules using BioSimSpace, run GPU-accelerated molecular dynamics via OpenMM, and then display the resulting dynamics trajectory in a NGLView Jupyter notebook 3D molecular viewer. This functionality is built on by BioSimSpace, which uses sire's molecular information engine to interconvert with programs such as GROMACS, NAMD, Amber, and AmberTools for automated molecular parameterization and the running of molecular dynamics, metadynamics, and alchemical free energy workflows. Sire comes complete with a powerful molecular information search engine, plus trajectory loading and editing, analysis, and energy evaluation engines. This, when combined with an in-built computer algebra system, gives substantial flexibility to researchers to load, search for, edit, and combine molecular information from multiple sources and use that to drive novel algorithms by combining functionality from other programs. Sire is open source (GPL3) and is available via conda and at a free Jupyter notebook server at https://try.openbiosim.org. Sire is supported by the not-for-profit OpenBioSim community interest company.
ABSTRACT
Resistance to antibiotics is a public health crisis. Although carbapenems are less susceptible to resistance than other ß-lactam antibiotics, ß-lactamases mediating resistance against these drugs are spreading. Here, we dissect the contributions of electric fields to carbapenemase activity in class A ß-lactamases. We perform QM/MM molecular dynamics simulations of meropenem acyl-enzyme hydrolysis that correctly discriminate carbapenemases. Electric field analysis shows that active-site fields in the deacylation transition state and tetrahedral intermediate are important determinants of activity. The active-site fields identify several residues, some distal, that distinguish efficient carbapenemases. Our field analysis script (www.github.com/bunzela/FieldTools) may help in understanding and combating antibiotic resistance.
ABSTRACT
Many enzymes display non-Arrhenius behavior with curved Arrhenius plots in the absence of denaturation. There has been significant debate about the origin of this behavior and recently the role of the activation heat capacity (ΔCP⧧) has been widely discussed. If enzyme-catalyzed reactions occur with appreciable negative values of ΔCP⧧ (arising from narrowing of the conformational space along the reaction coordinate), then curved Arrhenius plots are a consequence. To investigate these phenomena in detail, we have collected high precision temperature-rate data over a wide temperature interval for a model glycosidase enzyme MalL, and a series of mutants that change the temperature-dependence of the enzyme-catalyzed rate. We use these data to test a range of models including macromolecular rate theory (MMRT) and an equilibrium model. In addition, we have performed extensive molecular dynamics (MD) simulations to characterize the conformational landscape traversed by MalL in the enzyme-substrate complex and an enzyme-transition state complex. We have crystallized the enzyme in a transition state-like conformation in the absence of a ligand and determined an X-ray crystal structure at very high resolution (1.10 Å). We show (using simulation) that this enzyme-transition state conformation has a more restricted conformational landscape than the wildtype enzyme. We coin the term "transition state-like conformation (TLC)" to apply to this state of the enzyme. Together, these results imply a cooperative conformational transition between an enzyme-substrate conformation (ES) and a transition-state-like conformation (TLC) that precedes the chemical step. We present a two-state model as an extension of MMRT (MMRT-2S) that describes the data along with a convenient approximation with linear temperature dependence of the activation heat capacity (MMRT-1L) that can be used where fewer data points are available. Our model rationalizes disparate behavior seen for MalL and previous results for a thermophilic alcohol dehydrogenase and is consistent with a raft of data for other enzymes. Our model can be used to characterize the conformational changes required for enzyme catalysis and provides insights into the role of cooperative conformational changes in transition state stabilization that are accompanied by changes in heat capacity for the system along the reaction coordinate. TLCs are likely to be of wide importance in understanding the temperature dependence of enzyme activity and other aspects of enzyme catalysis.
ABSTRACT
Protein functions are dynamically regulated by allostery, which enables conformational communication even between faraway residues, and expresses itself in many forms, akin to different "languages": allosteric control pathways predominating in an unperturbed protein are often unintuitively reshaped whenever biochemical perturbations arise (e.g., mutations). To accurately model allostery, unbiased molecular dynamics (MD) simulations require integration with a reliable method able to, e.g., detect incipient allosteric changes or likely perturbation pathways; this is because allostery can operate at longer time scales than those accessible by plain MD. Such methods are typically applied singularly, but we here argue their joint applicationâas a "multilingual" approachâcould work significantly better. We successfully prove this through unbiased MD simulations (â¼100 µs) of the widely studied, allosterically active oncotarget K-Ras4B, solvated and embedded in a phospholipid membrane, from which we decrypt allostery using four showcase "languages": Distance Fluctuation analysis and the Shortest Path Map capture allosteric hotspots at equilibrium; Anisotropic Thermal Diffusion and Dynamical Non-Equilibrium MD simulations assess perturbations upon, respectively, either superheating or hydrolyzing the GTP that oncogenically activates K-Ras4B. Chosen "languages" work synergistically, providing an articulate, mutually coherent, experimentally consistent picture of K-Ras4B allostery, whereby distinct traits emerge at equilibrium and upon GTP cleavage. At equilibrium, combined evidence confirms prominent allosteric communication from the membrane-embedded hypervariable region, through a hub comprising helix α5 and sheet ß5, and up to the active site, encompassing allosteric "switches" I and II (marginally), and two proposed pockets. Upon GTP cleavage, allosteric perturbations mostly accumulate on the switches and documented interfaces.
Subject(s)
Molecular Dynamics Simulation , Proteins , Proteins/chemistry , Catalytic Domain , Guanosine Triphosphate/metabolism , Allosteric RegulationABSTRACT
Background: Due to COVID-19, pandemic preparedness emerges as a key imperative, necessitating new approaches to accelerate development of reagents against infectious pathogens. Methods: Here, we developed an integrated approach combining synthetic, computational and structural methods with in vitro antibody selection and in vivo immunization to design, produce and validate nature-inspired nanoparticle-based reagents against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Results: Our approach resulted in two innovations: (i) a thermostable nasal vaccine called ADDoCoV, displaying multiple copies of a SARS-CoV-2 receptor binding motif derived epitope and (ii) a multivalent nanoparticle superbinder, called Gigabody, against SARS-CoV-2 including immune-evasive variants of concern (VOCs). In vitro generated neutralizing nanobodies and electron cryo-microscopy established authenticity and accessibility of epitopes displayed by ADDoCoV. Gigabody comprising multimerized nanobodies prevented SARS-CoV-2 virion attachment with picomolar EC50. Vaccinating mice resulted in antibodies cross-reacting with VOCs including Delta and Omicron. Conclusion: Our study elucidates Adenovirus-derived dodecamer (ADDomer)-based nanoparticles for use in active and passive immunization and provides a blueprint for crafting reagents to combat respiratory viral infections.
ABSTRACT
We describe a two-step approach for combining interactive molecular dynamics in virtual reality (iMD-VR) with free energy (FE) calculation to explore the dynamics of biological processes at the molecular level. We refer to this combined approach as iMD-VR-FE. Stage one involves using a state-of-the-art 'human-in-the-loop' iMD-VR framework to generate a diverse range of protein-ligand unbinding pathways, benefitting from the sophistication of human spatial and chemical intuition. Stage two involves using the iMD-VR-sampled pathways as initial guesses for defining a path-based reaction coordinate from which we can obtain a corresponding free energy profile using FE methods. To investigate the performance of the method, we apply iMD-VR-FE to investigate the unbinding of a benzamidine ligand from a trypsin protein. The binding free energy calculated using iMD-VR-FE is similar for each pathway, indicating internal consistency. Moreover, the resulting free energy profiles can distinguish energetic differences between pathways corresponding to various protein-ligand conformations (e.g., helping to identify pathways that are more favourable) and enable identification of metastable states along the pathways. The two-step iMD-VR-FE approach offers an intuitive way for researchers to test hypotheses for candidate pathways in biomolecular systems, quickly obtaining both qualitative and quantitative insight.
Subject(s)
Proteins , Virtual Reality , Humans , Protein Binding , Ligands , Molecular Dynamics SimulationABSTRACT
Interactive molecular dynamics simulation in virtual reality (iMD-VR) is emerging as a promising technique in molecular science. Here, we demonstrate its use in a range of fifteen applications in materials science and heterogeneous catalysis. In this work, the iMD-VR package Narupa is used with the MD package, DL_POLY [1]. We show how iMD-VR can be used to: (i) investigate the mechanism of lithium fast ion conduction by directing the formation of defects showing that vacancy transport is favoured over interstitialcy mechanisms, and (ii) guide a molecule through a zeolite pore to explore diffusion within zeolites, examining in detail the motion of methyl n-hexanoate in H-ZSM-5 zeolite and identifying bottlenecks restricting diffusion. iMD-VR allows users to manipulate these systems intuitively, to drive changes in them and observe the resulting changes in structure and dynamics. We make these simulations available, as a resource for both teaching and research. All simulation files, with videos, can be found online (https://doi.org/10.5281/zenodo.8252314) and are provided as open-source material.
Subject(s)
Molecular Dynamics Simulation , Virtual Reality , Catalysis , Diffusion , Esters , LithiumABSTRACT
Owing to the emergence of antibiotic resistance, the polymyxin colistin has been recently revived to treat acute, multidrug-resistant Gram-negative bacterial infections. Positively charged colistin binds to negatively charged lipids and damages the outer membrane of Gram-negative bacteria. However, the MCR-1 protein, encoded by the mobile colistin resistance (mcr) gene, is involved in bacterial colistin resistance by catalysing phosphoethanolamine (PEA) transfer onto lipid A, neutralising its negative charge, and thereby reducing its interaction with colistin. Our preliminary results showed that treatment with a reference pyrazolone compound significantly reduced colistin minimal inhibitory concentrations in Escherichia coli expressing mcr-1 mediated colistin resistance (Hanpaibool et al. in ACS Omega, 2023). A docking-MD combination was used in an ensemble-based docking approach to identify further pyrazolone compounds as candidate MCR-1 inhibitors. Docking simulations revealed that 13/28 of the pyrazolone compounds tested are predicted to have lower binding free energies than the reference compound. Four of these were chosen for in vitro testing, with the results demonstrating that all the compounds tested could lower colistin MICs in an E. coli strain carrying the mcr-1 gene. Docking of pyrazolones into the MCR-1 active site reveals residues that are implicated in ligand-protein interactions, particularly E246, T285, H395, H466, and H478, which are located in the MCR-1 active site and which participate in interactions with MCR-1 in ≥ 8/10 of the lowest energy complexes. This study establishes pyrazolone-induced colistin susceptibility in E. coli carrying the mcr-1 gene, providing a method for the development of novel treatments against colistin-resistant bacteria.
Subject(s)
Escherichia coli Proteins , Pyrazolones , Colistin/pharmacology , Colistin/chemistry , Escherichia coli/metabolism , Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial/genetics , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Pyrazolones/pharmacology , Microbial Sensitivity TestsABSTRACT
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
Is scientific partnership between the UK and EU on the Horizon?
Subject(s)
European Union , United KingdomABSTRACT
Mutations in DNA gyrase confer resistance to fluoroquinolones, second-line antibiotics for Mycobacterium tuberculosis infections. Identification of new agents that inhibit M. tuberculosis DNA gyrase ATPase activity is one strategy to overcome this. Here, bioisosteric designs using known inhibitors as templates were employed to define novel inhibitors of M. tuberculosis DNA gyrase ATPase activity. This yielded the modified compound R3-13 with improved drug-likeness compared to the template inhibitor that acted as a promising ATPase inhibitor against M. tuberculosis DNA gyrase. Utilization of compound R3-13 as a virtual screening template, supported by subsequent biological assays, identified seven further M. tuberculosis DNA gyrase ATPase inhibitors with IC50 values in the range of 0.42-3.59 µM. The most active compound 1 showed an IC50 value of 0.42 µM, 3-fold better than the comparator ATPase inhibitor novobiocin (1.27 µM). Compound 1 showed noncytotoxicity to Caco-2 cells at concentrations up to 76-fold higher than its IC50 value. Molecular dynamics simulations followed by decomposition energy calculations identified that compound 1 occupies the binding pocket utilized by the adenosine group of the ATP analogue AMPPNP in the M. tuberculosis DNA gyrase GyrB subunit. The most prominent contribution to the binding of compound 1 to M. tuberculosis GyrB subunit is made by residue Asp79, which forms two hydrogen bonds with the OH group of this compound and also participates in the binding of AMPPNP. Compound 1 represents a potential new scaffold for further exploration and optimization as a M. tuberculosis DNA gyrase ATPase inhibitor and candidate anti-tuberculosis agent.