RESUMEN
The global increase in the morbidity/mortality rate of Mycobacterial infections, predominantly renascent tuberculosis, leprosy, and Buruli ulcers have become worrisome over the years. More challenging is the incidence of resistance mediated by mutant Mycobacterium strains against front-line antitubercular drugs. Homologous to all Mycobacteria species is the GlcNAc-6-phosphate deacetylase (NagA) which catalyzes essential amino sugars synthesis required for cell wall architecture, hence, metamorphosing into an important pharmacological target for curtailing virulence and drug-resistance. This study used integrated bioinformatics methods, MD simulations, and DynaMut and PolyPhen2 to; explore unique features, monitor dynamics, and analyze the functional impact of non-synonymous single-nucleotide polymorphisms of the six NagA of most ruinous Mycobacterium species; tuberculosis (Mtb), smegmatis (MS), marinum (MM), ulcerans, africanum, and microti respectively. This approach is essential for multi-targeting and could result in the identification of potential polypharmacological antitubercular compounds. Comparative sequential analyses revealed ≤ 50% of the overall structure, including the catalytic Asp267 and reactive Cys131, remained conserved. Interestingly, MS-NagA and MM-NagA possess unique hydrophobic isoleucine (Ile) residues at their active sites in contrast to leucine (Leu) found in other variants. More so, unique to the active sites of the NagA is a 'subunit loop' that covers the active site; probably crucial in binding (entry and exit) mechanisms of targeted NagA inhibitors. Relatively, nsSNP mutations exerted a destabilizing effect on the native NagA conformation. Structural and dynamical insights provided, basically pin-pointed the "Achilles' heel" explorable for the rational drug design of target-specific 'NagA' inhibitors potent against a wide range of mycobacterial diseases.
Asunto(s)
Amidohidrolasas/química , Proteínas Bacterianas/química , Mycobacterium tuberculosis/genética , Micobacterias no Tuberculosas/genética , Amidohidrolasas/genética , Proteínas Bacterianas/genética , Dominio Catalítico , Biología Computacional , Modelos Moleculares , Infecciones por Mycobacterium no Tuberculosas/microbiología , Polimorfismo de Nucleótido Simple , Conformación ProteicaRESUMEN
To successfully design and optimize the application of hydrogel matrices one has to effectively combine computational design tools with experimental methods. In this context, one of the most promising techniques is molecular modeling, which requires however accurate molecular models representing the investigated material. Although this method has been successfully used over the years for predicting the properties of polymers, its application to biopolymers, including gelatin, is limited. In this paper we provide a method for creating an atomistic representation of gelatin based on the modified FASTA codes of natural collagen. We show that the model created in this manner reproduces known experimental values of gelatin properties like density, glass-rubber transition temperature, WAXS profile and isobaric thermal expansion coefficient. We also present that molecular dynamics using the INTERFACE force field provides enough accuracy to track changes of density, fractional free volume and Hansen solubility coefficient over a narrow temperature regime (273-318 K) with 1 K accuracy. Thus we depict that using molecular dynamics one can predict properties of gelatin biopolymer as an efficient matrix for immobilization of various bioactive compounds, including enzymes.
Asunto(s)
Gelatina/química , Modelos Moleculares , Conformación Proteica , Algoritmos , Secuencia de Aminoácidos , Análisis de Varianza , Biopolímeros/química , Hidrogeles/química , TemperaturaRESUMEN
We characterized an operon in Mycobacterium tuberculosis, Rv3679-Rv3680, in which each open reading frame is annotated to encode "anion transporter ATPase" homologues. Using structure prediction modeling, we found that Rv3679 and Rv3680 more closely resemble the guided entry of tail-anchored proteins 3 (Get3) chaperone in eukaryotes. Get3 delivers proteins into the membranes of the endoplasmic reticulum and is essential for the normal growth and physiology of some eukaryotes. We sought to characterize the structures of Rv3679 and Rv3680 and test if they have a role in M. tuberculosis pathogenesis. We solved crystal structures of the nucleotide-bound Rv3679-Rv3680 complex at 2.5 to 3.2 Å and show that while it has some similarities to Get3 and ArsA, there are notable differences, including that these proteins are unlikely to be involved in anion transport. Deletion of both genes did not reveal any conspicuous growth defects in vitro or in mice. Collectively, we identified a new class of proteins in bacteria with similarity to Get3 complexes, the functions of which remain to be determined.IMPORTANCE Numerous bacterial species encode proteins predicted to have similarity with Get3- and ArsA-type anion transporters. Our studies provide evidence that these proteins, which we named BagA and BagB, are unlikely to be involved in anion transport. In addition, BagA and BagB are conserved in all mycobacterial species, including the causative agent of leprosy, which has a highly decayed genome. This conservation suggests that BagAB constitutes a part of the core mycobacterial genome and is needed for some yet-to-be-determined part of the life cycle of these organisms.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Animales , Proteínas de Transporte de Anión/genética , Femenino , Genoma Bacteriano , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/genética , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Operón , Unión Proteica , Conformación Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Ferric reductase B (FerB) is a flavin mononucleotide (FMN)-containing NAD(P)H:acceptor oxidoreductase structurally close to the Gluconacetobacter hansenii chromate reductase (ChrR). The crystal structure of ChrR was previously determined with a chloride bound proximal to FMN in the vicinity of Arg101, and the authors suggested that the anionic electron acceptors, chromate and uranyl tricarbonate, bind similarly. Here, we identify the corresponding arginine residue in FerB (Arg95) as being important for the reaction of FerB with superoxide. Four mutants at position 95 were prepared and found kinetically to have impaired capacity for superoxide binding. Stopped-flow data for the flavin cofactor showed that the oxidative step is rate limiting for catalytic turnover. The findings are consistent with a role for FerB as a superoxide scavenging contributor.
Asunto(s)
FMN Reductasa/química , Flavinas/genética , Conformación Proteica , Superóxidos/metabolismo , Secuencia de Aminoácidos/genética , Arginina/genética , Dominio Catalítico/genética , Cristalografía por Rayos X , FMN Reductasa/genética , Mononucleótido de Flavina/química , Mononucleótido de Flavina/genética , Flavinas/metabolismo , Cinética , Oxidación-Reducción , Oxidorreductasas/química , Oxidorreductasas/genética , Paracoccus denitrificans/química , Paracoccus denitrificans/enzimologíaRESUMEN
Of the more than 190 distinct species of Mycobacterium genus, many are economically and clinically important pathogens of humans or animals. Among those mycobacteria that infect humans, three species namely Mycobacterium tuberculosis (causative agent of tuberculosis), Mycobacterium leprae (causative agent of leprosy) and Mycobacterium abscessus (causative agent of chronic pulmonary infections) pose concern to global public health. Although antibiotics have been successfully developed to combat each of these, the emergence of drug-resistant strains is an increasing challenge for treatment and drug discovery. Here we describe the impact of the rapid expansion of genome sequencing and genome/pathway annotations that have greatly improved the progress of structure-guided drug discovery. We focus on the applications of comparative genomics, metabolomics, evolutionary bioinformatics and structural proteomics to identify potential drug targets. The opportunities and challenges for the design of drugs for M. tuberculosis, M. leprae and M. abscessus to combat resistance are discussed.
Asunto(s)
Proteínas Bacterianas/química , Biología Computacional/métodos , Mycobacterium/genética , Análisis de Secuencia de ADN/métodos , Animales , Proteínas Bacterianas/metabolismo , Descubrimiento de Drogas , Farmacorresistencia Bacteriana , Genoma Bacteriano , Humanos , Anotación de Secuencia Molecular , Mycobacterium/metabolismo , Mycobacterium abscessus/genética , Mycobacterium abscessus/metabolismo , Mycobacterium leprae/genética , Mycobacterium leprae/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Conformación Proteica , ProteómicaRESUMEN
Leprosy (causative, Mycobacterium leprae) continues to be the persisting public health problem with stable incidence rates, owing to the emergence of dapsone resistance that being the principal drug in the ongoing multidrug therapy. Hence, to overcome the drug resistance, structural modification through medicinal chemistry was used to design newer dapsone derivative(s) (DDs), against folic acid biosynthesis pathway. The approach included theoretical modeling, molecular docking, and molecular dynamic (MD) simulation as well as binding free energy estimation for validation of newly designed seven DDs, before synthesis. Theoretical modeling, docking, and MD simulation studies were used to understand the mode of binding and efficacy of DDs against the wild-type and mutant dihydropteroate synthases (DHPS). Principal component analysis was performed to understand the conformational dynamics of DHPS-DD complexes. Furthermore, the overall stability and negative-binding free energy of DHPS-DD complexes were deciphered using Molecular Mechanics/Poisson-Boltzmann Surface Area technique. Molecular mechanics study revealed that DD3 possesses higher binding free energy than dapsone against mutant DHPS. Energetic contribution analysis portrayed that van der Waals and electrostatic energy contributes profoundly to the overall negative free energy, whereas polar solvation energy opposes the binding. Finally, DD3 was synthesized and characterized using Fourier-transform infrared spectroscopy, UV, liquid chromatography-mass spectrometry, and proton nuclear magnetic resonance techniques. This study suggested that DD3 could be further promoted as newer antileprosy agent. The principles of medicinal chemistry and bioinformatics tools help to locate effective therapeutics to minimize resources and time in current drug development modules.
Asunto(s)
Dapsona/farmacología , Dihidropteroato Sintasa/antagonistas & inhibidores , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Mycobacterium leprae/enzimología , Dapsona/análogos & derivados , Dapsona/metabolismo , Dapsona/uso terapéutico , Dihidropteroato Sintasa/genética , Dihidropteroato Sintasa/metabolismo , Quimioterapia Combinada , Leprostáticos/farmacología , Leprostáticos/uso terapéutico , Mutación , Mycobacterium leprae/efectos de los fármacos , Unión Proteica , Conformación ProteicaRESUMEN
Cells possess protein quality control mechanisms to maintain proper cellular homeostasis. In eukaryotes, the roles of the ubiquitination and proteasome-mediated degradation of cellular proteins is well established. Recent studies have elucidated protein tagging mechanisms in prokaryotes, involving transfer messenger RNA (tmRNA) and pupylation. In this review, newer insights and bioinformatics analysis of two distinct bacterial protein tagging machineries are discussed. The machinery for tmRNAmediated tagging is present in several eubacterial representatives, e.g. Escherichia coli, Mycobacterium tuberculosis, Bacillus subtilis etc., but not in two archaeal representatives, such as Thermoplasma acidophilum and Sulfolobus solfataricus. On the other hand, the machinery involving tagging with the prokaryotic ubiquitin-like protein (Pup) is absent in most bacteria but is encoded in some eubacterial representatives, e.g. Mycobacterium tuberculosis and Mycobacterium leprae. Furthermore, molecular details on the relationship between protein tagging and enzymes involved in protein degradation in bacteria during infection are emerging. Several pathogenic bacteria that do not express the major ATP-dependent proteases, Lon and Caseinolytic protease (ClpP), are avirulent. Also, some ATP-independent peptidases, such as PepA and PepN, modulate the infection process. The roles of bacterial proteins involved in tagging and degradation during infection are discussed. These aspects add a new dimension to better understanding of the peculiarities of host-pathogen interactions.
Asunto(s)
Proteínas Arqueales/metabolismo , Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , ARN Bacteriano/metabolismo , Animales , Archaea/metabolismo , Proteínas Arqueales/genética , Bacterias/patogenicidad , Infecciones Bacterianas/microbiología , Proteínas Bacterianas/genética , Interacciones Huésped-Patógeno , Humanos , Péptido Hidrolasas/metabolismo , Conformación Proteica , Procesamiento Proteico-Postraduccional , Proteolisis , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
Photorhabdus asymbiotica is one of the three recognized species of the Photorhabdus genus, which consists of gram-negative bioluminescent bacteria belonging to the family Morganellaceae. These bacteria live in a symbiotic relationship with nematodes from the genus Heterorhabditis, together forming a complex that is highly pathogenic for insects. Unlike other Photorhabdus species, which are strictly entomopathogenic, P. asymbiotica is unique in its ability to act as an emerging human pathogen. Analysis of the P. asymbiotica genome identified a novel fucose-binding lectin designated PHL with a strong sequence similarity to the recently described P. luminescens lectin PLL. Recombinant PHL exhibited high affinity for fucosylated carbohydrates and the unusual disaccharide 3,6-O-Me2-Glcß1-4(2,3-O-Me2)Rhaα-O-(p-C6H4)-OCH2CH2NH2 from Mycobacterium leprae. Based on its crystal structure, PHL forms a seven-bladed ß-propeller assembling into a homo-dimer with an inter-subunit disulfide bridge. Investigating complexes with different ligands revealed the existence of two sets of binding sites per monomer-the first type prefers l-fucose and its derivatives, whereas the second type can bind d-galactose. Based on the sequence analysis, PHL could contain up to twelve binding sites per monomer. PHL was shown to interact with all types of red blood cells and insect haemocytes. Interestingly, PHL inhibited the production of reactive oxygen species induced by zymosan A in human blood and antimicrobial activity both in human blood, serum and insect haemolymph. Concurrently, PHL increased the constitutive level of oxidants in the blood and induced melanisation in haemolymph. Our results suggest that PHL might play a crucial role in the interaction of P. asymbiotica with both human and insect hosts.
Asunto(s)
Proteínas Bacterianas/inmunología , Interacciones Huésped-Patógeno/inmunología , Lectinas/inmunología , Photorhabdus/inmunología , Animales , Proteínas Bacterianas/genética , Secuencia de Bases , Cristalografía por Rayos X , Humanos , Lectinas/química , Lectinas/genética , Datos de Secuencia Molecular , Photorhabdus/genética , Conformación Proteica , Resonancia por Plasmón de SuperficieRESUMEN
It is becoming increasingly apparent that proteins are not static entities and that their function often critically depends on accurate sampling of multiple conformational states in aqueous solution. Accordingly, the development of methods to study conformational states in proteins beyond their ground-state structure ("excited states") has crucial biophysical importance. Here we investigate experimental schemes for optimally probing chemical exchange processes in proteins on the micro- to millisecond timescale by 15N R 1ρ relaxation dispersion. The schemes use selective Hartmann-Hahn cross-polarization (CP) transfer for excitation, and derive peak integrals from 1D NMR spectra (Korzhnev et al. in J Am Chem Soc 127:713-721, 2005; Hansen et al. in J Am Chem Soc 131:3818-3819, 2009). Simulation and experiment collectively show that in such CP-based schemes care has to be taken to achieve accurate suppression of undesired off-resonance coherences, when using weak spin-lock fields. This then (i) ensures that relaxation dispersion profiles in the absence of chemical exchange are flat, and (ii) facilitates extraction of relaxation dispersion profiles in crowded regions of the spectrum. Further improvement in the quality of the experimental data is achieved by recording the free-induction decays in an interleaved manner and including a heating-compensation element. The reported considerations will particularly benefit the use of CP-based R 1ρ relaxation dispersion to analyze conformational exchange processes in larger proteins, where resonance line overlap becomes the main limiting factor.
Asunto(s)
Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Proteínas/química , Marcaje Isotópico , Isótopos de NitrógenoRESUMEN
The transcription factor ScRpn4 coordinates the expression of Saccharomyces cerevisiae proteasomal genes. ScRpn4 orthologues are found in a number of other Saccharomycetes yeasts. Their functions, however, have not yet been characterised experimentally in vivo . We expressed the Debaryomyces hansenii DEHA2D12848 gene encoding an ScRpn4 orthologue (DhRpn4), in an S. cerevisiae strain lacking RPN4 . We showed that DhRpn4 activates transcription of proteasomal genes using ScRpn4 binding site and provides resistance to various stresses. The 43-238 aa segment of DhRpn4 contains an unique portable transactivation domain. Similar to the ScRpn4 N-terminus, this domain lacks a compact structure Moreover, upon overexpression in D. hansenii , DhRpn4 upregulates protesomal genes. Thus, we show that DhRpn4 is the activator for proteasomal genes.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Complejo de la Endopetidasa Proteasomal/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomycetales/enzimología , Factores de Transcripción/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Conformación Proteica , Dominios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Bacterial crystalline cellulose is used in biomedical and industrial applications, but the molecular mechanisms of synthesis are unclear. Unlike most bacteria, which make non-crystalline cellulose, Gluconacetobacter hansenii extrudes profuse amounts of crystalline cellulose. Its cellulose synthase (AcsA) exists as a complex with accessory protein AcsB, forming a 'terminal complex' (TC) that has been visualized by freeze-fracture TEM at the base of ribbons of crystalline cellulose. The catalytic AcsAB complex is embedded in the cytoplasmic membrane. The C-terminal portion of AcsC is predicted to form a translocation channel in the outer membrane, with the rest of AcsC possibly interacting with AcsD in the periplasm. It is thus believed that synthesis from an organized array of TCs coordinated with extrusion by AcsC and AcsD enable this bacterium to make crystalline cellulose. The only structural data that exist for this system are the above mentioned freeze-fracture TEM images, fluorescence microscopy images revealing that TCs align in a row, a crystal structure of AcsD bound to cellopentaose, and a crystal structure of PilZ domain of AcsA. Here we advance our understanding of the structural basis for crystalline cellulose production by bacterial cellulose synthase by determining a negative stain structure resolved to 23.4 Å for highly purified AcsAB complex that catalyzed incorporation of UDP-glucose into ß-1,4-glucan chains, and responded to the presence of allosteric activator cyclic diguanylate. Although the AcsAB complex was functional in vitro, the synthesized cellulose was not visible in TEM. The negative stain structure revealed that AcsAB is very similar to that of the BcsAB synthase of Rhodobacter sphaeroides, a non-crystalline cellulose producing bacterium. The results indicate that the crystalline cellulose producing and non-crystalline cellulose producing bacteria share conserved catalytic and membrane translocation components, and support the hypothesis that it is the extrusion mechanism and order in linearly arrayed TCs that enables production of crystalline cellulose.
Asunto(s)
Gluconacetobacter/enzimología , Glucosiltransferasas/química , Glucosiltransferasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Membrana Celular/metabolismo , Celulosa/biosíntesis , Cristalografía por Rayos X , Tomografía con Microscopio Electrónico , Gluconacetobacter/química , Modelos Moleculares , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Conformación ProteicaRESUMEN
Dapsone resistance is a serious impediment to the implementation of the present leprosy control strategies. In the recent past, many studies have been undertaken to address the antibiotic activity and binding pattern of dapsone against both native and mutant (Pro55Leu) folP1. Yet, there is no well-developed structural basis for understanding drug action and there is dire need for new antibacterial therapies. In the present study, molecular simulation techniques were employed alongside experimental strategies to address and overcome the mechanism of dapsone resistance. In essence, we report the identification of small molecule compounds to effectively and specifically inhibit the growth of M. leprae through targeting dihydropteroate synthase, encoded by folP1 which is involved in folic acid synthesis. Initially, ADME and toxicity studies were employed to screen the lead compounds, using dapsone as standard drug. Subsequently, molecular docking was employed to understand the binding efficiency of dapsone and its lead compounds against folP1. Further, the activity of the screened lead molecule was studied by means of molecular dynamics simulation techniques. Furthermore, we synthesized 4-(2-fluorophenylsulfonyl)benzenamine, using (2-fluorophenyl)boronic acid and 4-aminobenzenesulfonyl chloride, and the compound structure was confirmed by (1)H NMR and (13)C NMR spectroscopic techniques. Most importantly, the antibacterial activity of the compound was also examined and compared against dapsone. Overall, the result from our analysis suggested that CID21480113 (4-(2-fluorophenylsulfonyl)benzenamine) could be developed into a promising lead compound and could be effective in treating dapsone resistant leprosy cases.
Asunto(s)
Dapsona/farmacología , Dihidropteroato Sintasa/genética , Descubrimiento de Drogas , Farmacorresistencia Bacteriana , Leprostáticos/farmacología , Lepra/microbiología , Mutación , Mycobacterium leprae/efectos de los fármacos , Mycobacterium leprae/genética , Secuencia de Aminoácidos , Sitios de Unión , Dapsona/química , Dihidropteroato Sintasa/química , Humanos , Leprostáticos/química , Lepra/tratamiento farmacológico , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Unión Proteica , Conformación ProteicaRESUMEN
The molecular basis for determination of resistance to anti-leprosy drugs is the presence of point mutations within the genes of Mycobacterium leprae (M. leprae) that encode active drug targets. The downstream structural and functional implications of these point mutations on drug targets were scarcely studied. In this study, we utilized computational tools to develop native and mutant protein models for 5 point mutations at codon positions 53 and 55 in 6-hydroxymethyl-7, 8-dihydropteroate synthase (DHPS) of M. leprae, an active target for dapsone encoded by folp1 gene, that confer resistance to dapsone. Molecular docking was performed to identify variations in dapsone interaction with mutant DHPS in terms of hydrogen bonding, hydrophobic interactions, and energy changes. Schrodinger Suite 2014-3 was used to build homology models and in performing molecular docking. An increase in volume of the binding cavities of mutant structures was noted when compared to native form indicating a weakening in interaction (60.7 Å(3) in native vs. 233.6 Å(3) in Thr53Ala, 659.9 Å(3) in Thr53Ile, 400 Å(3) for Thr53Val, 385 Å(3) for Pro55Arg, and 210 Å(3) for Pro55Leu). This was also reflected by changes in hydrogen bonds and decrease in hydrophobic interactions in the mutant models. The total binding energy (ΔG) decreased significantly in mutant forms when compared to the native form (-51.92 Kcal/mol for native vs. -35.64, -35.24, -46.47, -47.69, and -41.36 Kcal/mol for mutations Thr53Ala, Thr53Ile, Thr53Val, Pro55Arg, and Pro55Leu, respectively. In brief, this analysis provided structural and mechanistic insights to the degree of dapsone resistance contributed by each of these DHPS mutants in leprosy.
Asunto(s)
Dapsona/administración & dosificación , Dihidropteroato Sintasa/química , Lepra/genética , Mycobacterium leprae/efectos de los fármacos , Dihidropteroato Sintasa/genética , Dihidropteroato Sintasa/metabolismo , Farmacorresistencia Bacteriana/genética , Humanos , Enlace de Hidrógeno , Lepra/tratamiento farmacológico , Simulación del Acoplamiento Molecular , Mycobacterium leprae/patogenicidad , Mutación Puntual , Unión Proteica , Conformación Proteica/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Adenosine-5'-triphosphate (ATP) is an important phosphate metabolite abundantly found in Mycobacterium leprae bacilli. This pathogen does not derive ATP from its host but has its own mechanism for the generation of ATP. Interestingly, this molecule as well as several antigenic proteins act as bio-markers for the detection of leprosy. One such bio-marker is the 18 kDa antigen. This 18 kDa antigen is a small heat shock protein (HSP18) whose molecular chaperone function is believed to help in the growth and survival of the pathogen. But, no evidences of interaction of ATP with HSP18 and its effect on the structure and chaperone function of HSP18 are available in the literature. Here, we report for the first time evidences of "HSP18-ATP" interaction and its consequences on the structure and chaperone function of HSP18. TNP-ATP binding experiment and surface plasmon resonance measurement showed that HSP18 interacts with ATP with a sub-micromolar binding affinity. Comparative sequence alignment between M. leprae HSP18 and αB-crystallin identified the sequence 49KADSLDIDIE58 of HSP18 as the Walker-B ATP binding motif. Molecular docking studies revealed that ß4-ß8 groove/strands as an ATP interactive region in M. leprae HSP18. ATP perturbs the tertiary structure of HSP18 mildly and makes it less susceptible towards tryptic cleavage. ATP triggers exposure of additional hydrophobic patches at the surface of HSP18 and induces more stability against chemical and thermal denaturation. In vitro aggregation and thermal inactivation assays clearly revealed that ATP enhances the chaperone function of HSP18. Our studies also revealed that the alteration in the chaperone function of HSP18 is reversible and is independent of ATP hydrolysis. As the availability and binding of ATP to HSP18 regulates its chaperone function, this functional inflection may play an important role in the survival of M. leprae in hosts.
Asunto(s)
Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Biomarcadores/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Mycobacterium leprae/metabolismo , Adenosina Trifosfato/análogos & derivados , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas de Choque Térmico/genética , Chaperonas Moleculares/genética , Mycobacterium leprae/fisiología , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Alineación de Secuencia , Resonancia por Plasmón de Superficie , Cadena B de alfa-Cristalina/genéticaRESUMEN
Homoserine dehydrogenase (HSD) from Mycobacterium leprae TN is an antifungal target for antifungal properties including efficacy against the human pathogen. The 3D structure of HSD has been firmly established by homology modeling methods. Using the template, homoserine dehydrogenase from Thiobacillus denitrificans (PDB Id 3MTJ), a sequence identity of 40% was found and molecular dynamics simulation was used to optimize a reliable structure. The substrate and co-factor-binding regions in HSD were identified. In order to determine the important residues of the substrate (L-aspartate semialdehyde (L-ASA)) binding, the ASA was docked to the protein; Thr163, Asp198, and Glu192 may be important because they form a hydrogen bond with HSD through AutoDock 4.2 software. neuraminidaseAfter use of a virtual screening technique of HSD, the four top-scoring docking hits all seemed to cation-π ion pair with the key recognition residue Lys107, and Lys207. These ligands therefore seemed to be new chemotypes for HSD. Our results may be helpful for further experimental investigations.
Asunto(s)
Inhibidores Enzimáticos/química , Homoserina Deshidrogenasa/química , Modelos Moleculares , Mycobacterium leprae/enzimología , Secuencia de Aminoácidos , Ácido Aspártico/análogos & derivados , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Sitios de Unión , Dominio Catalítico , Inhibidores Enzimáticos/metabolismo , Homoserina Deshidrogenasa/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , NAD/química , NAD/metabolismo , Conformación Proteica , Curva ROC , Reproducibilidad de los Resultados , Alineación de Secuencia , Especificidad por SustratoRESUMEN
The genus Mycobacterium is comprised of Gram-positive bacteria occupying a wide range of natural habitats and includes species that range from severe intracellular pathogens to economically useful and harmless microbes. The recent upsurge in the availability of microbial genome data has shown that genes encoding haemoglobin-like proteins are ubiquitous among Mycobacteria and that multiple haemoglobins (Hbs) of different classes may be present in pathogenic and non-pathogenic species. The occurrence of truncated haemoglobins (trHbs) and flavohaemoglobins (flavoHbs) showing distinct haem active site structures and ligand-binding properties suggests that these Hbs may be playing diverse functions in the cellular metabolism of Mycobacteria. TrHbs and flavoHbs from some of the severe human pathogens such as Mycobacterium tuberculosis and Mycobacterium leprae have been studied recently and their roles in effective detoxification of reactive nitrogen and oxygen species, electron cycling, modulation of redox state of the cell and facilitation of aerobic respiration have been proposed. This multiplicity in the function of Hbs may aid these pathogens to cope with various environmental stresses and survive during their intracellular regime. This chapter provides recent updates on genomic, structural and functional aspects of Mycobacterial Hbs to address their role in Mycobacteria.
Asunto(s)
Proteínas Bacterianas/metabolismo , Hemoproteínas/metabolismo , Mycobacterium/metabolismo , Hemoglobinas Truncadas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biotransformación , Hemoproteínas/química , Hemoproteínas/genética , Redes y Vías Metabólicas , Modelos Moleculares , Mycobacterium/química , Mycobacterium/genética , Óxido Nítrico/metabolismo , Óxido Nítrico/toxicidad , Oxidación-Reducción , Oxígeno/metabolismo , Oxígeno/toxicidad , Conformación Proteica , Especies de Nitrógeno Reactivo/metabolismo , Especies de Nitrógeno Reactivo/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Especies Reactivas de Oxígeno/toxicidad , Hemoglobinas Truncadas/química , Hemoglobinas Truncadas/genéticaRESUMEN
Binding of substrates into the active site, often through complementarity of shapes and charges, is central to the specificity of an enzyme. In many cases, substrate binding induces conformational changes in the active site, promoting specific interactions between them. In contrast, non-substrates either fail to bind or do not induce the requisite conformational changes upon binding and thus no catalysis occurs. In principle, both lock and key and induced-fit binding can provide specific interactions between the substrate and the enzyme. In this study, we present an interesting case where cofactor binding pre-tunes the active site geometry to recognize only the cognate substrates. We illustrate this principle by studying the substrate binding and kinetic properties of Xylose Reductase from Debaryomyces hansenii (DhXR), an AKR family enzyme which catalyzes the reduction of carbonyl substrates using NADPH as co-factor. DhXR reduces D-xylose with increased specificity and shows no activity towards "non-substrate" sugars like L-rhamnose. Interestingly, apo-DhXR binds to D-xylose and L-rhamnose with similar affinity (K(d)â¼5.0-10.0 mM). Crystal structure of apo-DhXR-rhamnose complex shows that L-rhamnose is bound to the active site cavity. L-rhamnose does not bind to holo-DhXR complex and thus, it cannot competitively inhibit D-xylose binding and catalysis even at 4-5 fold molar excess. Comparison of K(d) values with K(m) values reveals that increased specificity for D-xylose is achieved at the cost of moderately reduced affinity. The present work reveals a latent regulatory role for cofactor binding which was previously unknown and suggests that cofactor induced conformational changes may increase the complimentarity between D-xylose and active site similar to specificity achieved through induced-fit mechanism.
Asunto(s)
Aldehído Reductasa/metabolismo , Coenzimas/metabolismo , Proteínas Fúngicas/metabolismo , Holoenzimas/metabolismo , NADP/metabolismo , Saccharomycetales/enzimología , Xilosa/metabolismo , Aldehído Reductasa/química , Apoenzimas , Biocatálisis , Dominio Catalítico , Coenzimas/química , Cristalografía por Rayos X , Proteínas Fúngicas/química , Holoenzimas/química , Cinética , Modelos Moleculares , NADP/química , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ramnosa/química , Ramnosa/metabolismo , Saccharomycetales/química , Especificidad por Sustrato , Xilosa/químicaRESUMEN
Residue-to-alanine mutations and a two-amino acid deletion have been made in the highly conserved catalytic loop (residues 100-109) of Salmonella typhimurium OMP synthase (orotate phosphoribosyltransferase, EC 2.4.2.10). As described previously, the K103A mutant enzyme exhibited a 10(4)-fold decrease in k(cat)/K(M) for PRPP; the K100A enzyme suffered a 50-fold decrease. Alanine mutations at His105 and Glu107 produced 40- and 7-fold decreases in k(cat)/K(M), respectively, and E101A, D104A, and G106A were slightly faster than the wild-type (WT) in terms of k(cat), with minor effects on k(cat)/K(M). Equilibrium binding of OMP or PRPP in binary complexes was affected little by loop mutation, suggesting that the energetics of ground-state binding have little contribution from the catalytic loop, or that a favorable binding energy is offset by costs of loop reorganization. Pre-steady-state kinetics for mutants showed that K103A and E107A had lost the burst of product formation in each direction that indicated rapid on-enzyme chemistry for WT, but that the burst was retained by H105A. Δ102Δ106, a loop-shortened enzyme with Ala102 and Gly106 deleted, showed a 10(4)-fold reduction of k(cat) but almost unaltered K(D) values for all four substrate molecules. The 20% (i.e., 1.20) intrinsic [1'-(3)H]OMP kinetic isotope effect (KIE) for WT is masked because of high forward and reverse commitment factors. K103A failed to express intrinsic KIEs fully (1.095 ± 0.013). In contrast, H105A, which has a smaller catalytic lesion, gave a [1'-(3)H]OMP KIE of 1.21 ± 0.0005, and E107A (1.179 ± 0.0049) also gave high values. These results are interpreted in the context of the X-ray structure of the complete substrate complex for the enzyme [Grubmeyer, C., Hansen, M. R., Fedorov, A. A., and Almo, S. C. (2012) Biochemistry 51 (preceding paper in this issue, DOI 10.1021/bi300083p )]. The full expression of KIEs by H105A and E107A may result from a less secure closure of the catalytic loop. The lower level of expression of the KIE by K103A suggests that in these mutant proteins the major barrier to catalysis is successful closure of the catalytic loop, which when closed, produces rapid and reversible catalysis.
Asunto(s)
Orotato Fosforribosiltransferasa/genética , Orotato Fosforribosiltransferasa/metabolismo , Mutación Puntual , Salmonella typhimurium/enzimología , Alanina/química , Alanina/genética , Alanina/metabolismo , Dominio Catalítico , Cinética , Mutagénesis Sitio-Dirigida , Orotato Fosforribosiltransferasa/química , Conformación Proteica , Salmonella typhimurium/química , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismoRESUMEN
Leprosy is an infectious disease caused by Mycobacterium leprae. The increasing drug and multi-drug resistance of M. leprae enforce the importance of finding new drug targets. Mycobacterium has unusually impermeable cell wall that contributes to considerable resistance to many drugs. Peptidoglycan is an important component of the cell wall of M. leprae. UDP-N-acetylmuramoyl-glycyl-D-glutamate-2, 6-diaminopimelate ligase (MurE) plays a crucial role in the peptidoglycan biosynthesis and hence it could be considered as a potential drug target for leprosy. Structure of this enzyme for M. leprae has not yet been elucidated. We modeled the three-dimensional structure of MurE from M. leprae using comparative modeling methods based on the X-ray crystal structure of MurE from E. coli and validated. The 3D-structure of M. leprae MurE enzyme was docked with its substrates meso-diaminopimelic acid (A2pm) and UDP-N-acetyl muramoyl-glycyl-D- glutamate (UMGG) and its product UDP-N-acetyl muramoyl-glycyl-D-glu-meso-A(2)pm (UTP) and also with ATP. The docked complexes reveal the amino acids responsible for binding the substrates. Superposition of these complex structures suggests that carboxylic acid group of UMGG is positioned in proximity to γ-phosphate of the ATP to facilitate the formation of acylphosphate intermediate. The orientation of an amino group of A(2)pm facilitates the nucleophilic attack to form the product. Overall, the proposed model together with its binding features gained from docking studies could help to design a truly selective ligand inhibitor specific to MurE for the treatment of leprosy.
Asunto(s)
Modelos Moleculares , Mycobacterium leprae/enzimología , Péptido Sintasas/química , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Sitios de Unión , Simulación por Computador , Cristalografía por Rayos X , Diseño de Fármacos , Escherichia coli/enzimología , Modelos Químicos , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Alineación de Secuencia , Análisis de Secuencia de ProteínaRESUMEN
The crystal structures of two forms of Mycobacterium leprae single-stranded DNA-binding protein (SSB) have been determined at 2.05 and 2.8 Å resolution. Comparison of these structures with the structures of other eubacterial SSBs indicates considerable variation in their quaternary association, although the DNA-binding domains in all of them exhibit the same OB-fold. This variation has no linear correlation with sequence variation, but could be related to variation in protein stability. Molecular-dynamics simulations have been carried out on tetrameric molecules derived from the two forms and the prototype Escherichia coli SSB and the individual subunits of both proteins. Together, the X-ray studies and molecular-dynamics simulations yield information on the relatively rigid and flexible regions of the molecule and on the effect of oligomerization on flexibility. The simulations provide insight into the changes in subunit structure on oligomerization. They also provide insight into the stability and time evolution of the hydrogen bonds/water bridges that connect the two pairs of monomers in the tetramer.