Coarse-Grained Model of Glycosaminoglycans for Biomolecular Simulations.

Proteoglycans contain glycosaminoglycans (GAGs) which are negatively charged linear polymers made of repeating disaccharide units of uronic acid and hexosamine units. They play vital roles in numerous physiological and pathological processes, particularly in governing cellular communication and attachment. Depending on their sulfonation state, acetylation, and glycosidic linkages, GAGs belong to different families. The high molecular weight, heterogeneity, and flexibility of GAGs hamper their characterization at atomic resolution, but this may be circumvented via coarse-grained (CG) approaches. In this work, we report a CG model for a library of common GAG types in their isolated or proteoglycan-linked states compatible with version 2.2 (v2.2) of the widely popular CG Martini force field. The model reproduces conformational and thermodynamic properties for a wide variety of GAGs, as well as matching structural and binding data for selected proteoglycan test systems. The parameters developed here may thus be employed to study a range of GAG-containing biomolecular systems, thereby benefiting from the efficiency and broad applicability of the Martini framework.

High efficacy of the F-ATP synthase inhibitor TBAJ-5307 against nontuberculous mycobacteria in vitro and in vivo.

The F1FO-ATP synthase engine is essential for viability and growth of nontuberculous mycobacteria (NTM) by providing the biological energy ATP and keeping ATP homeostasis under hypoxic stress conditions. Here, we report the discovery of the diarylquinoline TBAJ-5307 as a broad spectrum anti-NTM inhibitor, targeting the FO domain of the engine and preventing rotation and proton translocation. TBAJ-5307 is active at low nanomolar concentrations against fast- and slow-growing NTM as well as clinical isolates by depleting intrabacterial ATP. As demonstrated for the fast grower Mycobacterium abscessus, the compound is potent in vitro and in vivo, without inducing toxicity. Combining TBAJ-5307 with anti-NTM antibiotics or the oral tebipenem-avibactam pair showed attractive potentiation. Furthermore, the TBAJ-5307-tebipenem-avibactam cocktail kills the pathogen, suggesting a novel oral combination for the treatment of NTM lung infections.

Variations of the Mycobacterium abscessus F-ATP synthase subunit a-c interface alter binding and potency of the anti-TB drug bedaquiline.

The anti-tuberculosis therapeutic bedaquiline (BDQ) is used against Mycobacterium abscessus. In M. abscessus BDQ is only bacteriostatic and less potent compared to M. tuberculosis or M. smegmatis. Here we demonstrate its reduced ATP synthesis inhibition against M. abscessus inside-out vesicles, including the F1FO-ATP synthase. Molecular dynamics simulations and binding free energy calculations highlight the differences in drug-binding of the M. abscessus and M. smegmatis FO-domain at the lagging site, where the drug deploys its mechanistic action, inhibiting ATP synthesis. These data pave the way for improved anti-M. abscessus BDQ analogs.

ATP binding by an F1Fo ATP synthase ε subunit is pH dependent, suggesting a diversity of ε subunit functional regulation in bacteria.

It is a conjecture that the ε subunit regulates ATP hydrolytic function of the F1Fo ATP synthase in bacteria. This has been proposed by the ε subunit taking an extended conformation, with a terminal helix probing into the central architecture of the hexameric catalytic domain, preventing ATP hydrolysis. The ε subunit takes a contracted conformation when bound to ATP, thus would not interfere with catalysis. A recent crystallographic study has disputed this; the Caldalkalibacillus thermarum TA2.A1 F1Fo ATP synthase cannot natively hydrolyse ATP, yet studies have demonstrated that the loss of the ε subunit terminal helix results in an ATP synthase capable of ATP hydrolysis, supporting ε subunit function. Analysis of sequence and crystallographic data of the C. thermarum F1Fo ATP synthase revealed two unique histidine residues. Molecular dynamics simulations suggested that the protonation state of these residues may influence ATP binding site stability. Yet these residues lie outside the ATP/Mg2+ binding site of the ε subunit. We then probed the effect of pH on the ATP binding affinity of the ε subunit from the C. thermarum F1Fo ATP synthase at various physiologically relevant pH values. We show that binding affinity changes 5.9 fold between pH 7.0, where binding is weakest, to pH 8.5 where it is strongest. Since the C. thermarum cytoplasm is pH 8.0 when it grows optimally, this correlates to the ε subunit being down due to ATP/Mg2+ affinity, and not being involved in blocking ATP hydrolysis. Here, we have experimentally correlated that the pH of the bacterial cytoplasm is of critical importance for ε subunit ATP affinity regulated by second-shell residues thus the function of the ε subunit changes with growth conditions.

Binding properties of the anti-TB drugs bedaquiline and TBAJ-876 to a mycobacterial F-ATP synthase.

Tuberculosis (TB), the deadly disease caused by Mycobacterium tuberculosis (Mtb), kills more people worldwide than any other bacterial infectious disease. There has been a recent resurgence of TB drug discovery activities, resulting in the identification of a number of novel enzyme inhibitors. Many of these inhibitors target the electron transport chain complexes and the F1FO-ATP synthase; these enzymes represent new target spaces for drug discovery, since the generation of ATP is essential for the bacterial pathogen's physiology, persistence, and pathogenicity. The anti-TB drug bedaquiline (BDQ) targets the Mtb F-ATP synthase and is used as salvage therapy against this disease. Medicinal chemistry efforts to improve the physio-chemical properties of BDQ resulted in the discovery of 3,5-dialkoxypyridine (DARQ) analogs to which TBAJ-876 belongs. TBAJ-876, a clinical development candidate, shows attractive in vitro and in vivo antitubercular activity. Both BDQ and TBAJ-876 inhibit the mycobacterial F1FO-ATP synthase by stopping rotation of the c-ring turbine within the FO domain, thereby preventing proton translocation and ATP synthesis to occur. While structural data for the BDQ bound state are available, no structural information about TBAJ-876 binding have been described. In this study, we show how TBAJ-876 binds to the FO domain of the M. smegmatis F1FO-ATP synthase. We further calculate the binding free energy of both drugs bound to their target and predict an increased affinity of TBAJ-876 for the FO domain. This approach will be useful in future efforts to design new and highly potent DARQ analogs targeting F-ATP synthases of Mtb, nontuberculosis mycobacteria (NTM) as well as the M. leprosis complex.

Bridging the N-terminal and middle domains in FliG of the flagellar rotor.

Flagella are necessary for bacterial movement and contribute to various aspects of virulence. They are complex cylindrical structures built of multiple molecular rings with self-assembly properties. The flagellar rotor is composed of the MS-ring and the C-ring. The FliG protein of the C-ring is central to flagellar assembly and function due to its roles in linking the C-ring with the MS-ring and in torque transmission from stator to rotor. No high-resolution structure of an assembled C-ring has been resolved to date, and the conformation adopted by FliG within the ring is unclear due to variations in available crystallographic data. Here, we use molecular dynamics (MD) simulations to study the conformation and dynamics of FliG in different states of assembly, including both in physiologically relevant and crystallographic lattice environments. We conclude that the linker between the FliG N-terminal and middle domain likely adopts an extended helical conformation in vivo, in contrast with the contracted conformation observed in some previous X-ray studies. We further support our findings with integrative model building of full-length FliG and a FliG ring model that is compatible with cryo-electron tomography (cryo-ET) and electron microscopy (EM) densities of the C-ring. Collectively, our study contributes to a better mechanistic understanding of the flagellar rotor assembly and its function.

A second shell residue modulates a conserved ATP-binding site with radically different affinities for ATP.

BACKGROUND: Prediction of ligand binding and design of new function in enzymes is a time-consuming and expensive process. Crystallography gives the impression that proteins adopt a fixed shape, yet enzymes are functionally dynamic. Molecular dynamics offers the possibility of probing protein movement while predicting ligand binding. Accordingly, we choose the bacterial F1Fo ATP synthase ε subunit to unravel why ATP affinity by ε subunits from Bacillus subtilis and Bacillus PS3 differs ~500-fold, despite sharing identical sequences at the ATP-binding site. METHODS: We first used the Bacillus PS3 ε subunit structure to model the B. subtilis ε subunit structure and used this to explore the utility of molecular dynamics (MD) simulations to predict the influence of residues outside the ATP binding site. To verify the MD predictions, point mutants were made and ATP binding studies were employed. RESULTS: MD simulations predicted that E102 in the B. subtilis ε subunit, outside of the ATP binding site, influences ATP binding affinity. Engineering E102 to alanine or arginine revealed a ~10 or ~54 fold increase in ATP binding, respectively, confirming the MD prediction that E102 drastically influences ATP binding affinity. CONCLUSIONS: These findings reveal how MD can predict how changes in the "second shell" residues around substrate binding sites influence affinity in simple protein structures. Our results reveal why seemingly identical ε subunits in different ATP synthases have radically different ATP binding affinities. GENERAL SIGNIFICANCE: This study may lead to greater utility of molecular dynamics as a tool for protein design and exploration of protein design and function.

An Alternative HIV-1 Non-Nucleoside Reverse Transcriptase Inhibition Mechanism: Targeting the p51 Subunit.

The ongoing development of drug resistance in HIV continues to push for the need of alternative drug targets in inhibiting HIV. One such target is the Reverse transcriptase (RT) enzyme which is unique and critical in the viral life cycle-a rational target that is likely to have less off-target effects in humans. Serendipitously, we found two chemical scaffolds from the National Cancer Institute (NCI) Diversity Set V that inhibited HIV-1 RT catalytic activity. Computational structural analyses and subsequent experimental testing demonstrated that one of the two chemical scaffolds binds to a novel location in the HIV-1 RT p51 subunit, interacting with residue Y183, which has no known association with previously reported drug resistance. This finding supports the possibility of a novel druggable site on p51 for a new class of non-nucleoside RT inhibitors that may inhibit HIV-1 RT allosterically. Although inhibitory activity was shown experimentally to only be in the micromolar range, the scaffolds serve as a proof-of-concept of targeting the HIV RT p51 subunit, with the possibility of medical chemistry methods being applied to improve inhibitory activity towards more effective drugs.

The Molecular Basis for Purine Binding Selectivity in the Bacterial ATP Synthase ϵ Subunit.

The ϵ subunit of ATP synthases has been proposed to regulate ATP hydrolysis in bacteria. Prevailing evidence supports the notion that when the ATP concentration falls below a certain threshold, the ϵ subunit changes its conformation from a non-inhibitory down-state to an extended up-state that then inhibits enzymatic ATP hydrolysis by binding to the catalytic domain. It has been demonstrated that the ϵ subunit from Bacillus PS3 is selective for ATP over other nucleotides, including GTP. In this study, the purine triphosphate selectivity is rationalized by using results from MD simulations and free energy calculations for the R103A/R115A mutant of the ϵ subunit from Bacillus PS3, which binds ATP more strongly than the wild-type protein. Our results are in good agreement with experimental data, and the elucidated molecular basis for selectivity could help to guide the design of novel GTP sensors.

Extending the Martini Coarse-Grained Force Field to N-Glycans.

Glycans play a vital role in a large number of cellular processes. Their complex and flexible nature hampers structure-function studies using experimental techniques. Molecular dynamics (MD) simulations can help in understanding dynamic aspects of glycans if the force field parameters used can reproduce key experimentally observed properties. Here, we present optimized coarse-grained (CG) Martini force field parameters for N-glycans, calibrated against experimentally derived binding affinities for lectins. The CG bonded parameters were obtained from atomistic (ATM) simulations for different glycan topologies including high mannose and complex glycans with various branching patterns. In the CG model, additional elastic networks are shown to improve maintenance of the overall conformational distribution. Solvation free energies and octanol-water partition coefficients were also calculated for various N-glycan disaccharide combinations. When using standard Martini nonbonded parameters, we observed that glycans spontaneously aggregated in the solution and required down-scaling of their interactions for reproduction of ATM model radial distribution functions. We also optimized the nonbonded interactions for glycans interacting with seven lectin candidates and show that a relatively modest scaling down of the glycan-protein interactions can reproduce free energies obtained from experimental studies. These parameters should be of use in studying the role of glycans in various glycoproteins and carbohydrate binding proteins as well as their complexes, while benefiting from the efficiency of CG sampling.

Characterizing the Hydration Properties of Proton Binding Sites in the ATP Synthase c-Rings of Bacillus Species.

The membrane-embedded domain of ATP synthases contains the c-ring, which translocates ions across the membrane, and its resultant rotation is coupled to ATP synthesis in the extramembranous domain. During rotation, the c-ring becomes accessible on both sides of the lipid bilayer to solvent via channels connected to the other membrane-embedded component, the a subunit, and thereby allows the ion to be released into the solvent environment. In recent times, many experimental structures of c-rings from different species have been solved. In some of these, a water molecule with a proposed "structural role" has been identified within the c-ring ion binding site, but in general, the requirement for high resolution to resolve specific water densities complicates their interpretation. In the present study, we use molecular dynamics (MD) simulations and rigorous free energy calculations to characterize the dynamics and energetics of a water molecule within the ion binding site of the c-ring from Bacillus pseudofirmus OF4, in its wild type (WT) and P51A mutant forms, along with the c-ring from thermophilic Bacillus PS3. Our data suggest that a water molecule stably binds to the P51A mutant, as well as helping to identify a bound water molecule in Bacillus PS3 whose presence was previously overlooked due to the limited resolution of the structural data. Sequence analysis further identifies a novel conserved sequence motif that is likely required to harbor a water molecule for stable ion coordination in the binding site of such proteins.

How Ligand Binding Affects the Dynamical Transition Temperature in Proteins.

The biochemical functions of proteins are activated at the protein glass transition temperature, which has been proposed to be dependent upon protein-water interactions. However, at the molecular level it is unclear how ligand binding to well-defined binding sites can influence this transition temperature. We thus report molecular dynamics (MD) simulations of the ϵ subunit from thermophilic Bacillus PS3 in the ATP-free and ligand-bound states over a range of temperatures from 20 to 300 K, to study the influence of ligand association upon the transition temperature. We also measure the protein mean square displacement (MSD) in each state, which is well established as a means to quantify this dynamical temperature dependence. We find that the transition temperature is largely unaffected by ligand association, but the MSD beyond the transition temperature increases more rapidly in the ATP-free state. Our data suggests that ligands can effectively "shield" a binding site from solvent, and hence stabilize protein domains with increasing temperature.

On the ion coupling mechanism of the MATE transporter ClbM.

Bacteria use a number of mechanisms to defend themselves from antimicrobial drugs. One important defense strategy is the ability to export drugs by multidrug transporters. One class of multidrug transporter, the so-called multidrug and toxic compound extrusion (MATE) transporters, extrude a variety of antibiotic compounds from the bacterial cytoplasm. These MATE transporters are driven by a Na+, H+, or combined Na+/H+ gradient, and act as antiporters to drive a conformational change in the transporter from the outward to the inward-facing conformation. In the inward-facing conformation, a chemical compound (drug) binds to the protein, resulting in a switch to the opposite conformation, thereby extruding the drug. Using molecular dynamics simulations, we now report the structural basis for Na+ and H+ binding in the dual ion coupled MATE transporter ClbM from Escherichia coli, which is connected to colibactin-induced genotoxicity, yielding novel insights into the ion/drug translocation mechanism of this bacterial transporter.

Insights into water accessible pathways and the inactivation mechanism of proton translocation by the membrane-embedded domain of V-type ATPases.

V-type ATPases are multi-protein complexes, which acidify cellular compartments in eukaryotes. They pump protons against an ion gradient, driven by a mechano-chemical framework that exploits ATP hydrolysis as an energy source. This process drives the rotation of the so-called c-ring, a membrane embedded complex in the Vo-domain of the V-type ATPase, resulting in translocation of protons across the membrane. One way in which the enzyme is regulated is by disassembly and reassembly of the V1-domain with the Vo-domain, which inactivates and reactivates the enzyme, respectively. Recently, structural data for the isolated Vo-domain from S. cerevisiae in an inactivated state were reported, suggesting the location of previously unobserved proton access pathways within the cytoplasmic and luminal compartments of the stator subunit a in Vo. However, the structural rationale for this inactivation remained unclear. In this study, the water accessibility pathway at the cytoplasmic side is confirmed, and novel insights into the role of the luminal channel with respect to the inactivation mechanism are obtained, using atomic-resolution molecular dynamics simulations. The results show that protonation of the key-glutamate, located in the c-ring of the Vo-domain, and facing the luminal compartment is preserved, when residing in the V1-depleted state. Maintaining the protonation of this essential glutamate is necessary to lock the luminal channel in the inactive, solvent-free state. Based on these theoretical observations and previous experimental results, a model of the proton translocation mechanism in the Vo-domain from V-type ATPases is proposed.

Single mutations in the ε subunit from thermophilic Bacillus PS3 generate a high binding affinity site for ATP.

The ε subunit from ATP synthases acts as an ATP sensor in the bacterial cell to prevent ATP hydrolysis and thus the waste of ATP under conditions of low ATP concentration. However, the ATP binding affinities from various bacterial organisms differ markedly, over several orders of magnitude. For example, the ATP synthases from thermophilic Bacillus PS3 and Escherichia coli exhibit affinities of 4 µM and 22 mM, respectively. The recently reported R103A/R115A double mutant of Bacillus PS3 ATP synthase demonstrated an increased binding affinity by two orders of magnitude with respect to the wild type. Here, we used atomic-resolution molecular dynamics simulations to determine the role of the R103A and R115A single mutations. These lead us to predict that both single mutations also cause an increased ATP binding affinity. Evolutionary analysis reveals R103 and R115 substitutions in the ε subunit from other bacillic organisms, leading us to predict they likely have a higher ATP binding affinity than previously expected.

Insights into the regulatory function of the ɛ subunit from bacterial F-type ATP synthases: a comparison of structural, biochemical and biophysical data.

ATP synthases catalyse the formation of ATP, the most common chemical energy storage unit found in living cells. These enzymes are driven by an electrochemical ion gradient, which allows the catalytic evolution of ATP by a binding change mechanism. Most ATP synthases are capable of catalysing ATP hydrolysis to varying degrees, and to prevent wasteful ATP hydrolysis, bacteria and mitochondria have regulatory mechanisms such as ADP inhibition. Additionally, ɛ subunit inhibition has also been described in three bacterial systems, Escherichia coli, Bacillus PS3 and Caldalkalibacillus thermarum TA2.A1. Previous studies suggest that the ɛ subunit is capable of undergoing an ATP-dependent conformational change from the ATP hydrolytic inhibitory 'extended' conformation to the ATP-induced non-inhibitory 'hairpin' conformation. A recently published crystal structure of the F1 domain of the C. thermarum TA2.A1 F1Fo ATP synthase revealed a mutant ɛ subunit lacking the ability to bind ATP in a hairpin conformation. This is a surprising observation considering it is an organism that performs no ATP hydrolysis in vivo, and appears to challenge the current dogma on the regulatory role of the ɛ subunit. This has prompted a re-examination of present knowledge of the ɛ subunits role in different organisms. Here, we compare published biochemical, biophysical and structural data involving ɛ subunit-mediated ATP hydrolysis regulation in a variety of organisms, concluding that the ɛ subunit from the bacterial F-type ATP synthases is indeed capable of regulating ATP hydrolysis activity in a wide variety of bacteria, making it a potentially valuable drug target, but its exact role is still under debate.

The structural basis of a high affinity ATP binding ε subunit from a bacterial ATP synthase.

The ε subunit from bacterial ATP synthases functions as an ATP sensor, preventing ATPase activity when the ATP concentration in bacterial cells crosses a certain threshold. The R103A/R115A double mutant of the ε subunit from thermophilic Bacillus PS3 has been shown to bind ATP two orders of magnitude stronger than the wild type protein. We use molecular dynamics simulations and free energy calculations to derive the structural basis of the high affinity ATP binding to the R103A/R115A double mutant. Our results suggest that the double mutant is stabilized by an enhanced hydrogen-bond network and fewer repulsive contacts in the ligand binding site. The inferred structural basis of the high affinity mutant may help to design novel nucleotide sensors based on the ε subunit from bacterial ATP synthases.

Insights into the ion-coupling mechanism in the MATE transporter NorM-VC.

Bacteria have developed a variety of different mechanisms to defend themselves from compounds that are toxic to them, such as antibiotics. One of these defence mechanisms is the expulsion of drugs or other noxious compounds by multidrug efflux pumps. Multidrug and toxic compound extrusion (MATE) transporters are efflux pumps that extrude metabolic waste and a variety of antibiotics out of the cell, using an ion gradient as energy source. They function via an alternating-access mechanism. When ions bind in the outward facing conformation, a large conformational change to the inward facing conformation is induced, from which the ion is released and the extruded chemical compound is bound. NorM proteins, which are usually coupled to a Na+ gradient, are members of the MATE family. However, for NorM-VC from Vibrio cholerae, it has been shown that this MATE transporter is additionally coupled to protons. How H+ and Na+ binding are coupled mechanistically to enable drug antiport is not well understood. In this study, we use molecular dynamics simulations to illuminate the sequence of ion binding events that enable efflux. Understanding this antiport mechanism is important to support the development of novel compounds that specifically inhibit the functional cycle of NorM transporters.

Predicted Structures of the Proton-Bound Membrane-Embedded Rotor Rings of the Saccharomyces cerevisiae and Escherichia coli ATP Synthases.

Recent years have witnessed a renewed interest in the ATP synthase as a drug target against human pathogens. Indeed, clinical, biochemical, and structural data indicate that hydrophobic inhibitors targeting the membrane-embedded proton-binding sites of the c-subunit ring could serve as last-resort antibiotics against multidrug resistant strains. However, because inhibition of the mitochondrial ATP synthase in humans is lethal, it is essential that these inhibitors be not only potent but also highly selective for the bacterial enzyme. To this end, a detailed understanding of the structure of this protein target is arguably instrumental. Here, we use computational methods to predict the atomic structures of the proton-binding sites in two prototypical c-rings: that of the ATP synthase from Saccharomyces cerevisiae, which is a model system for mitochondrial enzymes, and that from Escherichia coli, which can be pathogenic for humans. Our study reveals the structure of these binding sites loaded with protons and in the context of the membrane, that is, in the state that would mediate the recognition of a potential inhibitor. Both structures reflect a mode of proton coordination unlike those previously observed in other c-ring structures, whether experimental or modeled.

A role for loop G in the ß1 strand in GABAA receptor activation.

KEY POINTS: The role of the ß1 strand in GABAA receptor function is unclear. It lies anti-parallel to the ß2 strand, which is known to participate in receptor activation. Molecular dynamics simulation revealed solvent accessible residues within the ß1 strand of the GABAA ß3 homopentamer that might be amenable to analysis using the substituted Cys accessibility method. Cys substitutions from Asp43 to Thr47 in the GABAA α1 subunit showed that D43C and T47C reduced the apparent potency of GABA. F45C caused a biphasic GABA concentration-response relationship and increased spontaneous gating. Cys43 and Cys47 were accessible to 2-aminoethyl methanethiosulphonate (MTSEA) modification, whereas Cys45 was not. Both GABA and the allosteric agonist propofol reduced MTSEA modification of Cys43 and Cys47. By contrast, modification of Cys64 in the ß2 strand loop D was impeded by GABA but unaffected by propofol. These data reveal movement of ß1 strand loop G residues during agonist activation of the GABAA receptor. ABSTRACT: The GABAA receptor α subunit ß1 strand runs anti-parallel to the ß2 strand, which contains loop D, known to participate in receptor activation and agonist binding. However, a role for the ß1 strand has yet to be established. We used molecular dynamics simulation to quantify the solvent accessible surface area (SASA) of ß1 strand residues in the GABAA ß3 homopentamer structure. Residues in the complementary interface equivalent to those between Asp43 and Thr47 in the α1 subunit have an alternating pattern of high and low SASA consistent with a ß strand structure. We investigated the functional role of these ß1 strand residues in the α1 subunit by individually replacing them with Cys residues. D43C and T47C substitutions reduced the apparent potency of GABA at α1ß2γ2 receptors by 50-fold and eight-fold, respectively, whereas the F45C substitution caused a biphasic GABA concentration-response relationship and increased spontaneous gating. Receptors with D43C or T47C substitutions were sensitive to 2-aminoethyl methanethiosulphonate (MTSEA) modification. However, GABA-evoked currents mediated by α1(F45C)ß2γ2 receptors were unaffected by MTSEA, suggesting that this residue is inaccessible. Both GABA and the allosteric agonist propofol reduced MTSEA modification of α1(D43C)ß2γ2 and α1(T47C)ß2γ2 receptors, indicating movement of the ß1 strand even during allosteric activation. This is in contrast to α1(F64C)ß2γ2 receptors, where only GABA, but not propofol, reduced MTSEA modification. These findings provide the first functional evidence for movement of the ß1 strand during gating of the receptor and identify residues that are critical for maintaining GABAA receptor function.