ABSTRACT
Glucose transporters (GLUTs) are essential for organism-wide glucose homeostasis in mammals, and their dysfunction is associated with numerous diseases, such as diabetes and cancer. Despite structural advances, transport assays using purified GLUTs have proven to be difficult to implement, hampering deeper mechanistic insights. Here, we have optimized a transport assay in liposomes for the fructose-specific isoform GLUT5. By combining lipidomic analysis with native MS and thermal-shift assays, we replicate the GLUT5 transport activities seen in crude lipids using a small number of synthetic lipids. We conclude that GLUT5 is only active under a specific range of membrane fluidity, and that human GLUT1-4 prefers a similar lipid composition to GLUT5. Although GLUT3 is designated as the high-affinity glucose transporter, in vitro D-glucose kinetics demonstrates that GLUT1 and GLUT3 actually have a similar KM, but GLUT3 has a higher turnover. Interestingly, GLUT4 has a high KM for D-glucose and yet a very slow turnover, which may have evolved to ensure uptake regulation by insulin-dependent trafficking. Overall, we outline a much-needed transport assay for measuring GLUT kinetics and our analysis implies that high-levels of free fatty acid in membranes, as found in those suffering from metabolic disorders, could directly impair glucose uptake.
Subject(s)
Fatty Acids, Nonesterified , Liposomes , Humans , Animals , Kinetics , Glucose Transporter Type 1/genetics , Glucose Transporter Type 3/genetics , Glucose , MammalsABSTRACT
Visualizing ligand binding interactions is important for structure-based drug design and fragment-based screening methods. Rapid and uniform soaking with potentially reduced lattice defects make small macromolecular crystals attractive targets for studying drug binding using microcrystal electron diffraction (MicroED). However, so far no drug binding interactions could unambiguously be resolved by electron diffraction alone. Here, we use MicroED to study the binding of a sulfonamide inhibitor to human carbonic anhydrase isoform II (HCA II). We show that MicroED data can efficiently be collected on a conventional transmission electron microscope from thin hydrated microcrystals soaked with the clinical drug acetazolamide (AZM). The data are of high enough quality to unequivocally fit and resolve the bound inhibitor. We anticipate MicroED can play an important role in facilitating in-house fragment screening for drug discovery, complementing existing methods in structural biology such as X-ray and neutron diffraction.
Subject(s)
Acetazolamide/chemistry , Carbonic Anhydrase II/chemistry , Drug Evaluation, Preclinical , Microscopy, Electron, Transmission , Acetazolamide/therapeutic use , Carbonic Anhydrase II/antagonists & inhibitors , Crystallography, X-Ray , Electrons , Humans , Ligands , Pharmaceutical Preparations/chemistryABSTRACT
The decoration of secretory glycoproteins and glycolipids with sialic acid is critical to many physiological and pathological processes. Sialyation is dependent on a continuous supply of sialic acid into Golgi organelles in the form of CMP-sialic acid. Translocation of CMP-sialic acid into Golgi is carried out by the CMP-sialic acid transporter (CST). Mutations in human CST are linked to glycosylation disorders, and CST is important for glycopathway engineering, as it is critical for sialyation efficiency of therapeutic glycoproteins. The mechanism of how CMP-sialic acid is recognized and translocated across Golgi membranes in exchange for CMP is poorly understood. Here we have determined the crystal structure of a Zea mays CST in complex with CMP. We conclude that the specificity of CST for CMP-sialic acid is established by the recognition of the nucleotide CMP to such an extent that they are mechanistically capable of both passive and coupled antiporter activity.
Subject(s)
Cytidine Monophosphate N-Acetylneuraminic Acid/metabolism , Golgi Apparatus/metabolism , N-Acetylneuraminic Acid/metabolism , Nucleotide Transport Proteins/metabolism , Binding Sites , Crystallography, X-Ray , Humans , Models, Molecular , Nucleotide Transport Proteins/chemistry , Plant Proteins/chemistry , Plant Proteins/metabolism , Protein Conformation , Protein Multimerization , Zea mays/chemistry , Zea mays/metabolismABSTRACT
Crystal structures of two bacterial metal (Zn2+)-dependent d-fructose-1,6-bisphosphate (FBP) aldolases in complex with substrate, analogues, and triose-P reaction products were determined to 1.5-2.0 Å resolution. The ligand complexes cryotrapped in native or mutant Helicobacter pylori aldolase crystals enabled a novel mechanistic description of FBP C3-C4 bond cleavage. The reaction mechanism uses active site remodeling during the catalytic cycle, implicating relocation of the Zn2+ cofactor that is mediated by conformational changes of active site loops. Substrate binding initiates conformational changes triggered upon P1 phosphate binding, which liberates the Zn2+-chelating His-180, allowing it to act as a general base for the proton abstraction at the FBP C4 hydroxyl group. A second zinc-chelating His-83 hydrogen bonds the substrate C4 hydroxyl group and assists cleavage by stabilizing the developing negative charge during proton abstraction. Cleavage is concerted with relocation of the metal cofactor from an interior to a surface-exposed site, thereby stabilizing the nascent enediolate form. Conserved residue Glu-142 is essential for protonation of the enediolate form prior to product release. A d-tagatose 1,6-bisphosphate enzymatic complex reveals how His-180-mediated proton abstraction controls stereospecificity of the cleavage reaction. Recognition and discrimination of the reaction products, dihydroxyacetone-P and d-glyceraldehyde 3-P, occurs via charged hydrogen bonds between hydroxyl groups of the triose-Ps and conserved residues, Asp-82 and Asp-255, respectively, and are crucial aspects of the enzyme's role in gluconeogenesis. Conformational changes in mobile loops ß5-α7 and ß6-α8 (containing catalytic residues Glu-142 and His-180, respectively) drive active site remodeling, enabling the relocation of the metal cofactor.
Subject(s)
Catalytic Domain , Fructose-Bisphosphate Aldolase/metabolism , Fructose/metabolism , Helicobacter pylori/enzymology , Zinc/metabolism , Binding Sites , Catalysis , Crystallography, X-Ray , Fructose/chemistry , Fructose-Bisphosphate Aldolase/chemistry , Fructose-Bisphosphate Aldolase/genetics , Hydrogen Bonding , Models, Molecular , Mutation , Protein Conformation , Stereoisomerism , Substrate Specificity , Zinc/chemistryABSTRACT
At a time when the antibiotic drug discovery pipeline has stalled, antibiotic resistance is accelerating with catastrophic implications for our ability to treat bacterial infections. Globally we face the prospect of a future when common infections can once again kill. Anti-virulence approaches that target the capacity of the bacterium to cause disease rather than the growth or survival of the bacterium itself offer a tantalizing prospect of novel antimicrobials. They may also reduce the propensity to induce resistance by removing the strong selection pressure imparted by bactericidal or bacteriostatic agents. In the human pathogen Pseudomonas aeruginosa, disulfide bond protein A (PaDsbA1) plays a central role in the oxidative folding of virulence factors and is therefore an attractive target for the development of new anti-virulence antimicrobials. Using a fragment-based approach we have identified small molecules that bind to PaDsbA1. The fragment hits show selective binding to PaDsbA1 over the DsbA protein from Escherichia coli, suggesting that developing species-specific narrow-spectrum inhibitors of DsbA enzymes may be feasible. Structures of a co-complex of PaDsbA1 with the highest affinity fragment identified in the screen reveal that the fragment binds on the non-catalytic surface of the protein at a domain interface. This biophysical and structural data represent a starting point in the development of higher affinity compounds, which will be assessed for their potential as selective PaDsbA1 inhibitors.
Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Protein Disulfide-Isomerases/antagonists & inhibitors , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/drug effects , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Crystallography, X-Ray , Drug Discovery , Humans , Molecular Docking Simulation , Protein Binding , Protein Disulfide-Isomerases/chemistry , Protein Disulfide-Isomerases/metabolism , Pseudomonas Infections/drug therapy , Pseudomonas aeruginosa/chemistry , Pseudomonas aeruginosa/metabolism , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Virulence Factors/metabolismABSTRACT
Sodium/proton exchangers of the SLC9 family mediate the transport of protons in exchange for sodium to help regulate intracellular pH, sodium levels, and cell volume. In electrogenic Na+/H+ antiporters, it has been assumed that two ion-binding aspartate residues transport the two protons that are later exchanged for one sodium ion. However, here we show that we can switch the antiport activity of the bacterial Na+/H+ antiporter NapA from being electrogenic to electroneutral by the mutation of a single lysine residue (K305). Electroneutral lysine mutants show similar ion affinities when driven by [Formula: see text]pH, but no longer respond to either an electrochemical potential ([Formula: see text]) or could generate one when driven by ion gradients. We further show that the exchange activity of the human Na+/H+ exchanger NHA2 (SLC9B2) is electroneutral, despite harboring the two conserved aspartic acid residues found in NapA and other bacterial homologues. Consistently, the equivalent residue to K305 in human NHA2 has been replaced with arginine, which is a mutation that makes NapA electroneutral. We conclude that a transmembrane embedded lysine residue is essential for electrogenic transport in Na+/H+ antiporters.
Subject(s)
Antiporters/metabolism , Sodium-Hydrogen Exchangers/metabolism , Thermus thermophilus/metabolism , Amino Acid Sequence , Amino Acid Substitution , Antiporters/chemistry , Aspartic Acid/chemistry , Bacteria/metabolism , Binding Sites , Cysteine/chemistry , Electrochemistry , Humans , Hydrogen-Ion Concentration , Ion Transport , Lysine/chemistry , Models, Molecular , Mutagenesis, Site-Directed , Protein Conformation , Protons , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Sodium/metabolism , Species SpecificityABSTRACT
Na+/H+ antiporters are found in all kingdoms of life and exhibit catalysis rates that are among the fastest of all known secondary-active transporters. Here we combine ion mobility mass spectrometry and molecular dynamics simulations to study the conformational stability and lipid-binding properties of the Na+/H+ exchanger NapA from Thermus thermophilus and compare this to the prototypical antiporter NhaA from Escherichia coli and the human homologue NHA2. We find that NapA and NHA2, but not NhaA, form stable dimers and do not selectively retain membrane lipids. By comparing wild-type NapA with engineered variants, we show that the unfolding of the protein in the gas phase involves the disruption of inter-domain contacts. Lipids around the domain interface protect the native fold in the gas phase by mediating contacts between the mobile protein segments. We speculate that elevator-type antiporters such as NapA, and likely NHA2, use a subset of annular lipids as structural support to facilitate large-scale conformational changes within the membrane.
Subject(s)
Lipids/chemistry , Mass Spectrometry/methods , Sodium-Hydrogen Exchangers/chemistry , Sodium-Hydrogen Exchangers/metabolism , Antiporters/chemistry , Antiporters/genetics , Antiporters/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Lipid Metabolism , Molecular Dynamics Simulation , Protein Domains , Protein Stability , Protein Unfolding , Sodium-Hydrogen Exchangers/genetics , Thermus thermophilus/chemistryABSTRACT
To fully understand the transport mechanism of Na(+)/H(+) exchangers, it is necessary to clearly establish the global rearrangements required to facilitate ion translocation. Currently, two different transport models have been proposed. Some reports have suggested that structural isomerization is achieved through large elevator-like rearrangements similar to those seen in the structurally unrelated sodium-coupled glutamate-transporter homolog GltPh. Others have proposed that only small domain movements are required for ion exchange, and a conventional rocking-bundle model has been proposed instead. Here, to resolve these differences, we report atomic-resolution structures of the same Na(+)/H(+) antiporter (NapA from Thermus thermophilus) in both outward- and inward-facing conformations. These data combined with cross-linking, molecular dynamics simulations and isothermal calorimetry suggest that Na(+)/H(+) antiporters provide alternating access to the ion-binding site by using elevator-like structural transitions.
Subject(s)
Sodium-Hydrogen Exchangers/chemistry , Sodium-Hydrogen Exchangers/metabolism , Thermus thermophilus/enzymology , Calorimetry , Crystallography, X-Ray , Models, Molecular , Molecular Dynamics Simulation , Protein ConformationABSTRACT
Pseudomonas aeruginosa is an opportunistic human pathogen for which new antimicrobial drug options are urgently sought. P. aeruginosa disulfide-bond protein A1 (PaDsbA1) plays a pivotal role in catalyzing the oxidative folding of multiple virulence proteins and as such holds great promise as a drug target. As part of a fragment-based lead discovery approach to PaDsbA1 inhibitor development, the identification of a crystal form of PaDsbA1 that was more suitable for fragment-soaking experiments was sought. A previously identified crystallization condition for this protein was unsuitable, as in this crystal form of PaDsbA1 the active-site surface loops are engaged in the crystal packing, occluding access to the target site. A single residue involved in crystal-packing interactions was substituted with an amino acid commonly found at this position in closely related enzymes, and this variant was successfully used to generate a new crystal form of PaDsbA1 in which the active-site surface is more accessible for soaking experiments. The PaDsbA1 variant displays identical redox character and in vitro activity to wild-type PaDsbA1 and is structurally highly similar. Two crystal structures of the PaDsbA1 variant were determined in complex with small molecules bound to the protein active site. These small molecules (MES, glycerol and ethylene glycol) were derived from the crystallization or cryoprotectant solutions and provide a proof of principle that the reported crystal form will be amenable to co-crystallization and soaking with small molecules designed to target the protein active-site surface.
Subject(s)
Alkanesulfonic Acids/chemistry , Bacterial Proteins/chemistry , Ethylene Glycol/chemistry , Glycerol/chemistry , Morpholines/chemistry , Protein Disulfide-Isomerases/chemistry , Pseudomonas aeruginosa/chemistry , Amino Acid Sequence , Amino Acid Substitution , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Catalytic Domain , Crystallography, X-Ray , Gene Expression , Models, Molecular , Molecular Sequence Data , Mutation , Protein Binding , Protein Disulfide-Isomerases/genetics , Protein Disulfide-Isomerases/metabolism , Protein Engineering , Protein Structure, Secondary , Pseudomonas aeruginosa/enzymology , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Structural Homology, ProteinABSTRACT
The altered activity of the fructose transporter GLUT5, an isoform of the facilitated-diffusion glucose transporter family, has been linked to disorders such as type 2 diabetes and obesity. GLUT5 is also overexpressed in certain tumour cells, and inhibitors are potential drugs for these conditions. Here we describe the crystal structures of GLUT5 from Rattus norvegicus and Bos taurus in open outward- and open inward-facing conformations, respectively. GLUT5 has a major facilitator superfamily fold like other homologous monosaccharide transporters. On the basis of a comparison of the inward-facing structures of GLUT5 and human GLUT1, a ubiquitous glucose transporter, we show that a single point mutation is enough to switch the substrate-binding preference of GLUT5 from fructose to glucose. A comparison of the substrate-free structures of GLUT5 with occluded substrate-bound structures of Escherichia coli XylE suggests that, in addition to global rocker-switch-like re-orientation of the bundles, local asymmetric rearrangements of carboxy-terminal transmembrane bundle helices TM7 and TM10 underlie a 'gated-pore' transport mechanism in such monosaccharide transporters.
Subject(s)
Fructose/metabolism , Glucose Transporter Type 5/chemistry , Glucose Transporter Type 5/metabolism , Animals , Binding Sites , Biological Transport , Cattle , Cell Membrane/metabolism , Crystallography, X-Ray , Escherichia coli/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Fructose/chemistry , Glucose/chemistry , Glucose/metabolism , Glucose Transporter Type 1/chemistry , Glucose Transporter Type 1/metabolism , Glucose Transporter Type 5/genetics , Models, Molecular , Point Mutation/genetics , Protein Conformation , Rats , Salts/chemistry , Static Electricity , Structure-Activity Relationship , Substrate Specificity/genetics , Symporters/chemistry , Symporters/metabolismABSTRACT
Mass spectrometry of intact membrane protein complexes requires removal of the detergent micelle by collisional activation. We demonstrate that the necessary energy can be obtained by adjusting the degree of collisional cooling in the ion source. This enables us to extend the energy regime for dissociation of membrane protein complexes.
Subject(s)
Membrane Proteins/chemistry , Thermodynamics , Gases/chemistry , Mass Spectrometry , Protein UnfoldingABSTRACT
UNLABELLED: Epstein-Barr virus (EBV) is the etiologic agent of infectious mononucleosis and the root cause of B-cell lymphoproliferative disease in individuals with a weakened immune system, as well as a principal cofactor in nasopharyngeal carcinoma, various lymphomas, and other cancers. The EBV major virion surface glycoprotein gp350 is viewed as the best vaccine candidate to prevent infectious mononucleosis in healthy EBV-naive persons and EBV-related cancers in at-risk individuals. Previous epitope mapping of gp350 revealed only one dominant neutralizing epitope, which has been shown to be the target of the monoclonal antibody 72A1. Computer modeling of the 72A1 antibody interaction with the gp350 amino terminus was used to identify gp350 amino acids that could form strong ionic, electrostatic, or hydrogen bonds with the 72A1 antibody. Peptide DDRTTLQLAQNPVYIPETYPYIKWDN (designated peptide 2) and peptide GSAKPGNGSYFASVKTEMLGNEID (designated peptide 3) were designed to spatially represent the gp350 amino acids predicted to interact with the 72A1 antibody paratope. Peptide 2 bound to the 72A1 antibody and blocked 72A1 antibody recognition of the native gp350 molecule. Peptide 2 and peptide 3 were recognized by human IgG and shown to elicit murine antibodies that could target gp350 and block its recognition by the 72A1 antibody. This work provides a structural mapping of the interaction between the EBV-neutralizing antibody 72A1 and the major virion surface protein gp350. gp350 mimetic peptides that spatially depict the EBV-neutralizing epitope would be useful as a vaccine to focus the immune system exclusively to this important virus epitope. IMPORTANCE: The production of virus-neutralizing antibodies targeting the Epstein-Barr virus (EBV) major surface glycoprotein gp350 is important for the prevention of infectious mononucleosis and EBV-related cancers. The data presented here provide the first in silico map of the gp350 interaction with a virus-blocking monoclonal antibody. Immunization with gp350 peptides identified by in silico mapping generated antibodies that cross-react with the EBV gp350 molecule and block recognition of the gp350 molecule by a virus-neutralizing antibody. Through its ability to focus the immune system exclusively on the gp350 sequence important for viral entry, these peptides may form the basis of an EBV vaccine candidate. This strategy would sidestep the production of other irrelevant gp350 antibodies that divert the immune system from generating a protective antiviral response or that impede access to the virus-blocking epitope by protective antibodies.
Subject(s)
Antibodies, Anti-Idiotypic/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Epitopes/immunology , Herpesvirus 4, Human/immunology , Peptides/immunology , Viral Proteins/immunology , Animals , DNA, Viral/chemistry , DNA, Viral/genetics , Female , Mice, Inbred BALB C , Molecular Sequence Data , Sequence Analysis, DNAABSTRACT
Crystal structures of divalent metal-dependent pyruvate aldolase, HpaI, in complex with substrate and cleavage products were determined to 1.8-2.0 Å resolution. The enzyme·substrate complex with 4-hydroxy-2-ketoheptane-1,7-dioate indicates that water molecule W2 bound to the divalent metal ion initiates C3-C4 bond cleavage. The binding mode of the aldehyde donor delineated a solvent-filled capacious binding locus lined with predominantly hydrophobic residues. The absence of direct interactions with the aldehyde aliphatic carbons accounts for the broad specificity and lack of stereospecific control by the enzyme. Enzymatic complex structures formed with keto acceptors, pyruvate, and 2-ketobutyrate revealed bidentate interaction with the divalent metal ion by C1-carboxyl and C2-carbonyl oxygens and water molecule W4 that is within close contact of the C3 carbon. Arg(70) assumes a multivalent role through its guanidinium moiety interacting with all active site enzymatic species: C2 oxygen in substrate, pyruvate, and ketobutyrate; substrate C4 hydroxyl; aldehyde C1 oxygen; and W4. The multiple interactions made by Arg(70) stabilize the negatively charged C4 oxygen following proton abstraction, the aldehyde alignment in aldol condensation, and the pyruvate enolate upon aldol cleavage as well as support proton exchange at C3. This role is corroborated by loss of aldol cleavage ability and pyruvate C3 proton exchange activity and by a 730-fold increase in the dissociation constant toward the pyruvate enolate analog oxalate in the R70A mutant. Based on the crystal structures, a mechanism is proposed involving the two enzyme-bound water molecules, W2 and W4, in acid/base catalysis that facilitates reversible aldol cleavage. The same reaction mechanism promotes decarboxylation of oxaloacetate.
Subject(s)
Acetoacetates/chemistry , Aldehyde-Lyases/chemistry , Multienzyme Complexes/chemistry , Pyruvic Acid/chemistry , Acetoacetates/metabolism , Aldehyde-Lyases/metabolism , Animals , Crystallography, X-Ray , Humans , Multienzyme Complexes/metabolism , Protein Structure, Quaternary , Pyruvic Acid/metabolism , RabbitsABSTRACT
Secretion systems translocate virulence factors of many bacterial pathogens, enabling their survival inside the host organism. Consequently, inhibition strongly attenuates pathogenicity and can be considered a target for novel antimicrobial drugs. The type IV secretion system (T4SS) of the intracellular pathogen Brucella is a prerequisite for its virulence, and in this work we targeted the interactions of the essential assembly factor protein, VirB8, using small-molecule inhibitors. High-throughput screening identified several potent and specific inhibitors, and the target-binding site of these inhibitors was identified by X-ray crystallography, in silico docking, and analysis of the derivates of the inhibitor B8I-2. VirB8 interaction inhibitors bind to a surface groove opposite to the dimerization interface, and by varying the binding-site residues, we were able to determine which residues are required for inhibitor activity. E115 and K182 were found to be especially important, and changes at R114, Y229, and L151 also reduced inhibitor efficiency.
Subject(s)
Bacterial Proteins/antagonists & inhibitors , Brucella/metabolism , Virulence Factors/antagonists & inhibitors , Amino Acid Substitution , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Computer Simulation , Crystallography, X-Ray , Dimerization , High-Throughput Screening Assays , Kinetics , Protein Structure, Tertiary , Structure-Activity Relationship , Virulence Factors/genetics , Virulence Factors/metabolismABSTRACT
The search for antituberculosis drugs active against persistent bacilli has led to our interest in metallodependent class II fructose-1,6-bisphosphate aldolase (FBA-tb), a key enzyme of gluconeogenesis absent from mammalian cells. Knock-out experiments at the fba-tb locus indicated that this gene is required for the growth of Mycobacterium tuberculosis on gluconeogenetic substrates and in glucose-containing medium. Surface labeling and enzymatic activity measurements revealed that this enzyme was exported to the cell surface of M. tuberculosis and produced under various axenic growth conditions including oxygen depletion and hence by non-replicating bacilli. Importantly, FBA-tb was also produced in vivo in the lungs of infected guinea pigs and mice. FBA-tb bound human plasmin(ogen) and protected FBA-tb-bound plasmin from regulation by α(2)-antiplasmin, suggestive of an involvement of this enzyme in host/pathogen interactions. The crystal structures of FBA-tb in the native form and in complex with a hydroxamate substrate analog were determined to 2.35- and 1.9-Å resolution, respectively. Whereas inhibitor attachment had no effect on the plasminogen binding activity of FBA-tb, it competed with the natural substrate of the enzyme, fructose 1,6-bisphosphate, and substantiated a previously unknown reaction mechanism associated with metallodependent aldolases involving recruitment of the catalytic zinc ion by the substrate upon active site binding. Altogether, our results highlight the potential of FBA-tb as a novel therapeutic target against both replicating and non-replicating bacilli.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Fructose-Bisphosphate Aldolase/chemistry , Fructose-Bisphosphate Aldolase/metabolism , Gluconeogenesis , Mycobacterium tuberculosis/enzymology , Tuberculosis, Pulmonary/enzymology , Animals , Bacterial Proteins/genetics , Crystallography, X-Ray , Fibrinolysin/genetics , Fibrinolysin/metabolism , Fructose-Bisphosphate Aldolase/genetics , Fructosediphosphates/chemistry , Fructosediphosphates/genetics , Fructosediphosphates/metabolism , Gene Knockdown Techniques , Guinea Pigs , Host-Pathogen Interactions/genetics , Humans , Mice , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/pathogenicity , Protein Binding , Tuberculosis, Pulmonary/genetics , alpha-2-Antiplasmin/genetics , alpha-2-Antiplasmin/metabolismABSTRACT
We report the synthesis and biochemical evaluation of several selective inhibitors of class II (zinc dependent) fructose bis-phosphate aldolases (Fba). The products were designed as transition-state analogues of the catalyzed reaction, structurally related to the substrate fructose bis-phosphate (or sedoheptulose bis-phosphate) and based on an N-substituted hydroxamic acid, as a chelator of the zinc ion present in active site. The compounds synthesized were tested on class II Fbas from various pathogenic microorganisms and, by comparison, on a mammalian class I Fba. The best inhibitor shows K(i) against class II Fbas from various pathogens in the nM range, with very high selectivity (up to 10(5)). Structural analyses of inhibitors in complex with aldolases rationalize and corroborate the enzymatic kinetics results. These inhibitors represent lead compounds for the preparation of new synthetic antibiotics, notably for tuberculosis prophylaxis.
Subject(s)
Anti-Bacterial Agents/chemical synthesis , Antifungal Agents/chemical synthesis , Fructose-Bisphosphate Aldolase/antagonists & inhibitors , Hydroxamic Acids/chemical synthesis , Zinc/physiology , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Antifungal Agents/chemistry , Antifungal Agents/pharmacology , Antitubercular Agents/chemical synthesis , Antitubercular Agents/chemistry , Antitubercular Agents/pharmacology , Crystallography, X-Ray , Drug Design , Fructosediphosphates/chemistry , Hydroxamic Acids/chemistry , Hydroxamic Acids/pharmacology , Inhibitory Concentration 50 , Kinetics , Microbial Sensitivity Tests , Models, Molecular , Rabbits , Structure-Activity Relationship , Sugar Phosphates/chemistryABSTRACT
Apicomplexan parasites rely on actin-based motility to drive host cell invasion. Prior in vitro studies implicated aldolase, a tetrameric glycolytic enzyme, in coupling actin filaments to the parasite's surface adhesin microneme protein 2 (MIC2). Here, we test the essentiality of this interaction in host cell invasion. Based on in vitro studies and homology modeling, we generated a series of mutations in Toxoplasma gondii aldolase (TgALD1) that delineated MIC2 tail domain (MIC2t) binding function from its enzyme activity. We tested these mutants by complementing a conditional knockout of TgALD1. Mutations that affected glycolysis also reduced motility. Mutants only affecting binding to MIC2t had no motility phenotype, but were decreased in their efficiency of host cell invasion. Our studies demonstrate that aldolase is not only required for energy production but is also essential for efficient host cell invasion, based on its ability to bridge adhesin-cytoskeleton interactions in the parasite.
Subject(s)
Cell Adhesion , Energy Metabolism , Fructose-Bisphosphate Aldolase/metabolism , Membrane Proteins/metabolism , Protein Interaction Mapping , Protozoan Proteins/metabolism , Toxoplasma/physiology , Actins/metabolism , Animals , Cell Line , Fructose-Bisphosphate Aldolase/genetics , Genetic Complementation Test , Host-Parasite Interactions , Models, Molecular , Mutation , Protein Structure, Tertiary , Protozoan Proteins/genetics , VirulenceABSTRACT
We report the synthesis and biochemical evaluation of selective inhibitors of class II (zinc-dependent) fructose bisphosphate aldolases. The most active compound is a simplified analogue of fructose bisphosphate, bearing a well-positioned metal chelating group. It is a powerful and highly selective competitive inhibitor of isolated class II aldolases. We report crystallographic studies of this inhibitor bound in the active site of the Helicobacter pylori enzyme. The compound also shows activity against Mycobacterium tuberculosis isolates.
Subject(s)
Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacology , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Fructose-Bisphosphate Aldolase/antagonists & inhibitors , Crystallography , Microbial Sensitivity Tests , Models, Molecular , Mycobacterium tuberculosis/drug effectsABSTRACT
An irreversible competitive inhibitor hydroxynaphthaldehyde phosphate was synthesized that is highly selective against the glycolytic enzyme fructose 1,6-bisphosphate aldolase from Trypanosoma brucei (causative agent of sleeping sickness). Inhibition involves Schiff base formation by the inhibitor aldehyde with Lys116 followed by reaction of the resultant Schiff base with a second residue. Molecular simulations indicate significantly greater molecular geometries conducive for nucleophilic attack in T. brucei aldolase than the mammalian isozyme and suggest Ser48 as the Schiff base modifying residue.
Subject(s)
Aldehydes/chemical synthesis , Fructose-Bisphosphate Aldolase/antagonists & inhibitors , Fructose-Bisphosphate Aldolase/chemistry , Naphthols/chemical synthesis , Organophosphates/chemical synthesis , Trypanocidal Agents/chemical synthesis , Trypanosoma brucei brucei/enzymology , Aldehydes/chemistry , Animals , Kinetics , Models, Molecular , Naphthols/chemistry , Organophosphates/chemistry , Schiff Bases/chemistry , Trypanocidal Agents/chemistryABSTRACT
Protein classifications show that the Rossmann fold, which consists of two betaalphabetaalphabeta motifs (BABAB) related by a rough twofold axis, is the most populated alphabeta fold, and that the betaalphabeta submotif (BAB) is a widespread elementary structural arrangement. Herein, we report MD simulations, circular dichroism and NMR analyses on BAB and BABAB from porcine lactate dehydrogenase to evaluate their intrinsic stability. Our results demonstrate that BAB is not stable in solution and is not a folding nucleus. We also find that BABAB, despite its appearance of a functional and structural unit, is not an independent and thermodynamically stable folding unit. Rather, we show that BABAB retains most native secondary structure but very little tertiary structure, thus displaying characteristics of a molten globule.