RESUMEN
The transition from mitotic to meiotic cell cycles is essential for haploid gamete formation and fertility. Stimulated by retinoic acid gene 8 (Stra8) is an essential gatekeeper of meiotic initiation in vertebrates; yet, the molecular role of STRA8 remains principally unknown. Here we demonstrate that STRA8 functions as a suppressor of autophagy during spermatogenesis in mice. Stra8-deficient germ cells fail to enter meiosis and present aberrant upregulation of autophagy-lysosome genes, commensurate with autophagy activation. Biochemical assays show that ectopic expression of STRA8 alone is sufficient to inhibit both autophagy induction and maturation. Studies also revealed that, Nr1d1, a nuclear hormone receptor gene, is upregulated in Stra8-deficient testes and that STRA8 binds to the Nr1d1 promoter, indicating that Nr1d1 is a direct target of STRA8 transcriptional repression. In addition, it was found that NR1D1 binds to the promoter of Ulk1, a gene essential for autophagy initiation, and that Nr1d1 is required for the upregulated Ulk1 expression in Stra8-deficient testes. Furthermore, both genetic deletion of Nr1d1 and pharmacologic inhibition of NR1D1 by its synthetic antagonist SR8278 exhibit rescuing effects on the meiotic initiation defects observed in Stra8-deficient male germ cells. Together, the data suggest a novel link between STRA8-mediated autophagy suppression and meiotic initiation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Fertilidad/genética , Meiosis , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/genética , Espermatogénesis/genética , Espermatozoides/metabolismo , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Animales , Autofagia/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Secuencia de Bases , Regulación del Desarrollo de la Expresión Génica , Isoquinolinas/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/antagonistas & inhibidores , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Espermatozoides/citología , Testículo/citología , Testículo/crecimiento & desarrollo , Testículo/metabolismo , Tiofenos/farmacologíaRESUMEN
We have previously reported benzimidazole-based compounds to be potent inhibitors of FabI for Francisella tularensis (FtFabI), making them promising antimicrobial hits. Optically active enantiomers exhibit markedly differing affinities toward FtFabI. The IC50 of benzimidazole (-)-1 is â¼100× lower than the (+)-enantiomer, with similar results for the 2 enantiomers. Determining the absolute configuration for these optical compounds and elucidating their binding modes is important for further design. Electronic circular dichroism (ECD) quantum calculations have become important in determining absolute configurations of optical compounds. We determined the absolute configuration of (-)/(+)-1 and (-)/(+)-2 by comparing experimental spectra and theoretical density functional theory (DFT) simulations of ECD spectra at the B3LYP/6-311+G(2d, p) level using Gaussian09. Comparison of experimental and calculated ECD spectra indicates that the S configuration corresponds to the (-)-rotation for both compounds 1 and 2, while the R configuration corresponds to the (+)-rotation. Further, molecular dynamics simulations and MM-GBSA binding energy calculations for these two pairs of enantiomers with FtFabI show much tighter binding MM-GBSA free energies for S-1 and S-2 than for their enantiomers, R-1 and R-2, consistent with the S configuration being the more active one, and with the ECD determination of the S configuration corresponding to (-) and the R configuration corresponding to (+). Thus, our computational studies allow us to assign (-) to (S)- and (+) to (R)- for compounds 1 and 2, and to further evaluate structural changes to improve efficacy.
Asunto(s)
Antibacterianos/farmacología , Bencimidazoles/farmacología , Enoil-CoA Hidratasa/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Francisella tularensis/efectos de los fármacos , Teoría Cuántica , Antibacterianos/química , Bencimidazoles/química , Sitios de Unión/efectos de los fármacos , Dicroismo Circular , Relación Dosis-Respuesta a Droga , Enoil-CoA Hidratasa/metabolismo , Inhibidores Enzimáticos/química , Francisella tularensis/enzimología , Enlace de Hidrógeno , Pruebas de Sensibilidad Microbiana , Simulación de Dinámica Molecular , Estructura Molecular , Relación Estructura-ActividadRESUMEN
The de novo purine biosynthesis pathway is an attractive target for antibacterial drug design, and PurE from this pathway has been identified to be crucial for Bacillus anthracis survival in serum. In this study we adopted a fragment-based hit discovery approach, using three screening methods-saturation transfer difference nucleus magnetic resonance (STD-NMR), water-ligand observed via gradient spectroscopy (WaterLOGSY) NMR, and surface plasmon resonance (SPR), against B. anthracis PurE (BaPurE) to identify active site binding fragments by initially testing 352 compounds in a Zenobia fragment library. Competition STD NMR with the BaPurE product effectively eliminated non-active site binding hits from the primary hits, selecting active site binders only. Binding affinities (dissociation constant, KD) of these compounds varied between 234 and 301µM. Based on test results from the Zenobia compounds, we subsequently developed and applied a streamlined fragment screening strategy to screen a much larger library consisting of 3000 computationally pre-selected fragments. Thirteen final fragment hits were confirmed to exhibit binding affinities varying from 14µM to 700µM, which were categorized into five different basic scaffolds. All thirteen fragment hits have ligand efficiencies higher than 0.30. We demonstrated that at least two fragments from two different scaffolds exhibit inhibitory activity against the BaPurE enzyme.
Asunto(s)
Bacillus anthracis/enzimología , Inhibidores Enzimáticos/farmacología , Transferasas Intramoleculares/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Dominio Catalítico/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Ensayos Analíticos de Alto Rendimiento , Transferasas Intramoleculares/aislamiento & purificación , Transferasas Intramoleculares/metabolismo , Espectroscopía de Resonancia Magnética , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Resonancia por Plasmón de SuperficieRESUMEN
4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleotide synthetase (PurC) is a key enzyme in the de novo purine biosynthetic pathway of bacteria and an ideal target pathway for the discovery of antimicrobials. Bacillus anthracis (Ba) and Streptococcus pneumoniae (Sp) are two of the bacteria shown to be severe detriments to public health. To be able to carry out the experimentation that leads to drug discovery, high yields of pure soluble recombinant protein must first be obtained. We studied two recombinant PurC proteins from B. anthracis and S. pneumoniae, using Escherichia coli as the host cells. These two proteins, with very similar amino acid sequences, exhibit very different solution properties, leading to a large difference in yields during protein purification under the same conditions. The yield for SpPurC (>50mG per gram of cells) is ten times greater than that for BaPurC (<5mG per gram of cells). The BaPurC samples in solution consisted of oligomers and dimers, with dimers as its functional form. Comparing the yields of dimers, SpPurC is 25 times greater than that for BaPurC (â¼2mG per gram of cell). Our studies suggest that the difference in exposed hydrophobic surface area is responsible for the difference in yields under the same conditions.
Asunto(s)
Bacillus anthracis/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Péptido Sintasas/química , Péptido Sintasas/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Streptococcus pneumoniae/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Interacciones Hidrofóbicas e Hidrofílicas , Datos de Secuencia Molecular , Péptido Sintasas/genética , Péptido Sintasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , SolubilidadRESUMEN
N(5)-carboxy-amino-imidazole ribonucleotide (N(5)-CAIR) mutase (PurE), a bacterial enzyme in the de novo purine biosynthetic pathway, has been suggested to be a target for antimicrobial agent development. We have optimized a thermal shift method for high-throughput screening of compounds binding to Bacillus anthracis PurE. We used a low ionic strength buffer condition to accentuate the thermal shift stabilization induced by compound binding to Bacillus anthracis PurE. The compounds identified were then subjected to computational docking to the active site to further select compounds likely to be inhibitors. A UV-based enzymatic activity assay was then used to select inhibitory compounds. Minimum inhibitory concentration (MIC) values were subsequently obtained for the inhibitory compounds against Bacillus anthracis (ΔANR strain), Escherichia coli (BW25113 strain, wild-type and ΔTolC), Francisella tularensis, Staphylococcus aureus (both methicillin susceptible and methicillin-resistant strains) and Yersinia pestis. Several compounds exhibited excellent (0.05-0.15µg/mL) MIC values against Bacillus anthracis. A common core structure was identified for the compounds exhibiting low MIC values. The difference in concentrations for inhibition and MIC suggest that another enzyme(s) is also targeted by the compounds that we identified.
Asunto(s)
Antiinfecciosos/farmacología , Bacillus anthracis/efectos de los fármacos , Bacillus anthracis/enzimología , Inhibidores Enzimáticos/farmacología , Antiinfecciosos/química , Inhibidores Enzimáticos/química , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Structures of (N(5))-carboxyaminoimidazole ribonucleotide synthase (PurK) from Bacillus anthracis with various combinations of ATP, ADP, Mg(2+), bicarbonate and aminoimidazole ribonucleotide (AIR) in the active site are presented. The binding site of bicarbonate has only been speculated upon previously, but is shown here for the first time. The binding involves interactions with the conserved residues Arg272, His274 and Lys348. These structures provide insights into each ligand in the active site and allow a possible mechanism to be proposed for the reaction that converts bicarbonate and AIR, in the presence of ATP, to produce (N(5))-carboxyaminoimidazole ribonucleotide. The formation of a carboxyphosphate intermediate through ATP phosphoryl transfer is proposed, followed by carboxylation of AIR to give the product, facilitated by a cluster of conserved residues and an active-site water network.
Asunto(s)
Bacillus anthracis/enzimología , Bicarbonatos/metabolismo , Ligasas/química , Ligasas/metabolismo , Adenosina Trifosfato/metabolismo , Bacillus anthracis/química , Bacillus anthracis/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Imidazoles/metabolismo , Magnesio/metabolismo , Modelos Moleculares , Ribonucleótidos/metabolismoRESUMEN
Streptococcus pneumoniae is a multidrug-resistant pathogen that is a target of considerable interest for antibacterial drug development. One strategy for drug discovery is to inhibit an essential metabolic enzyme. The seventh step of the de novo purine-biosynthesis pathway converts carboxyaminoimidazoleribonucleotide (CAIR) and L-aspartic acid (Asp) to 4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleotide (SAICAR) in the presence of adenosine 5'-triphosphate (ATP) using the enzyme PurC. PurC has been shown to be conditionally essential for bacterial replication. Two crystal structures of this essential enzyme from Streptococcus pneumoniae (spPurC) in the presence of adenosine 5'-diphosphate (ADP), Mg(2+), aminoimidazoleribonucleotide (AIR) and/or Asp have been obtained. This is the first structural study of spPurC, as well as the first of PurC from any species with Asp in the active site. Based on these findings, two model structures are proposed for the active site with all of the essential ligands (ATP, Mg(2+), Asp and CAIR) present, and a relay mechanism for the formation of the product SAICAR is suggested.
Asunto(s)
Adenosina Difosfato/química , Ácido Aspártico/química , Proteínas Bacterianas/química , Magnesio/química , Péptido Sintasas/química , Ribonucleótidos/química , Streptococcus pneumoniae/enzimología , Vías Biosintéticas , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Ligandos , Unión Proteica , Especificidad por SustratoRESUMEN
Extraordinary geogenic concentrations of cadmium (Cd) have been reported for some Jamaican soils. However, the bioavailability of the metal in these soils remains unknown. Here, the bioavailability of Cd in selected Jamaican soils was investigated through the determination of total and sequentially extractable concentrations in paired soil-plant (yam; Dioscorea sp.) samples (n = 24), using neutron activation analysis and atomic absorption spectroscopy as primary analytical techniques. Our results indicate that total soil Cd varied widely (2.2-148.7 mg kg(-1)), and on average, total extractable Cd accounted for ~55 % of the total soil Cd. The exchangeable and oxidizable species averaged 1.5 and 6.4 % of the total Cd, respectively, and, based on Spearman analysis, are the best predictors of yam Cd. There is also good evidence to suggest that variation in the bioavailability of the metal is in part controlled by the geochemical characteristics of the soils analyzed and is best explained by pH, cation exchange capacity (CEC) and organic matter content (% LOI).
Asunto(s)
Cadmio/análisis , Monitoreo del Ambiente , Contaminantes del Suelo/análisis , Suelo/química , JamaicaRESUMEN
We present an approach that accelerates protein solid-state NMR 5-20-fold using paramagnetic doping to condense data-collection time (to approximately 0.2 s per scan), overcoming a long-standing limitation on slow recycling owing to intrinsic (1)H T(1) longitudinal spin relaxation. Using low-power schemes under magic-angle spinning at 40 kHz, we obtained two-dimensional (13)C-(13)C and (13)C-(15)N solid-state NMR spectra for several to tens of nanomoles of beta-amyloid fibrils and ubiquitin in 1-2 d.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Amiloide/química , Péptidos beta-Amiloides/química , Isótopos de Carbono/química , Ácido Edético/química , Humanos , Isótopos de Nitrógeno/química , Fragmentos de Péptidos/química , Conformación Proteica , Espectrina/química , Ubiquitina/químicaRESUMEN
We have solved the crystal structure of a segment of nonerythroid alpha-spectrin (alphaII) consisting of the first 147 residues to a resolution of 2.3 A. We find that the structure of this segment is generally similar to a corresponding segment from erythroid alpha-spectrin (alphaI) but exhibits unique differences with functional significance. Specific features include the following: (i) an irregular and frayed first helix (Helix C'); (ii) a helical conformation in the junction region connecting Helix C' with the first structural domain (D1); (iii) a long A(1)B(1) loop in D1; and (iv) specific inter-helix hydrogen bonds/salt bridges that stabilize D1. Our findings suggest that the hydrogen bond networks contribute to structural domain stability, and thus rigidity, in alphaII, and the lack of such hydrogen bond networks in alphaI leads to flexibility in alphaI. We have previously shown the junction region connecting Helix C' to D1 to be unstructured in alphaI (Park, S., Caffrey, M. S., Johnson, M. E., and Fung, L. W. (2003) J. Biol. Chem. 278, 21837-21844) and now find it to be helical in alphaII, an important difference for alpha-spectrin association with beta-spectrin in forming tetramers. Homology modeling and molecular dynamics simulation studies of the structure of the tetramerization site, a triple helical bundle of partial domain helices, show that mutations in alpha-spectrin will affect Helix C' structural flexibility and/or the junction region conformation and may alter the equilibrium between spectrin dimers and tetramers in cells. Mutations leading to reduced levels of functional tetramers in cells may potentially lead to abnormal neuronal functions.
Asunto(s)
Encéfalo/metabolismo , Células Eritroides/metabolismo , Multimerización de Proteína , Proteínas Recombinantes/química , Espectrina/química , Cristalización , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Mutación/genética , Estructura Secundaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrina/genética , Espectrina/metabolismoRESUMEN
With yeast two-hybrid methods, we used a C-terminal fragment (residues 1697-2145) of non-erythroid beta spectrin (ßII-C), including the region involved in the association with alpha spectrin to form tetramers, as the bait to screen a human brain cDNA library to identify proteins interacting with ßII-C. We applied stringent selection steps to eliminate false positives and identified 17 proteins that interacted with ßII-C (IP(ßII-C) s). The proteins include a fragment (residues 38-284) of "THAP domain containing, apoptosis associated protein 3, isoform CRA g", "glioma tumor suppressor candidate region gene 2" (residues 1-478), a fragment (residues 74-442) of septin 8 isoform c, a fragment (residues 704-953) of "coatomer protein complex, subunit beta 1, a fragment (residues 146-614) of zinc-finger protein 251, and a fragment (residues 284-435) of syntaxin binding protein 1. We used yeast three-hybrid system to determine the effects of these ßII-C interacting proteins as well as of 7 proteins previously identified to interact with the tetramerization region of non-erythroid alpha spectrin (IP(αII-N) s) [1] on spectrin tetramer formation. The results showed that 3 IP(ßII-C) s were able to bind ßII-C even in the presence of αII-N, and 4 IP(αII-N) s were able to bind αII-N in the presence of ßII-C. We also found that the syntaxin binding protein 1 fragment abolished αII-N and ßII-C interaction, suggesting that this protein may inhibit or regulate non-erythroid spectrin tetramer formation.
Asunto(s)
Encéfalo/metabolismo , Proteínas Portadoras/metabolismo , Citoesqueleto/metabolismo , Proteínas de Microfilamentos/metabolismo , Neuronas/metabolismo , Fragmentos de Péptidos/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Sitios de Unión , Encéfalo/citología , Proteínas Portadoras/química , Proteínas Portadoras/genética , Línea Celular , Citoesqueleto/química , Biblioteca de Genes , Humanos , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/genética , Modelos Moleculares , Neuronas/citología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Plásmidos , Polimerizacion , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
Yeast two-hybrid (Y2H) and isothermal titration calorimetry (ITC) methods were used to further study the mutational effect of non-erythroid alpha spectrin (αII) at position 22 in tetramer formation with beta spectrin (ßII). Four mutants, αII-V22D, V22F, V22M and V22W, were studied. For the Y2H system, we used plasmids pGBKT7, consisting of the cDNA of the first 359 residues at the N-terminal region of αII, and pGADT7, consisting of the cDNA of residues 1697-2145 at the C-terminal region of ßII. Strain AH109 yeast cells were used for colony growth assays and strain Y187 was used for ß-galactosidase activity assays. Y2H results showed that the C-terminal region of ßII interacts with the N-terminal region of αII, either the wild type, or those with V22F, V22M or V22W mutations. The V22D mutant did not interact with ßII. For ITC studies, we used recombinant proteins of the αII N-terminal fragment and of the erythroid beta spectrin (ßI) C-terminal fragment; results showed that the K(d) values for V22F were similar to those for the wild-type (about 7 nM), whereas the K(d) values were about 35 nM for V22M and about 90 nM for V22W. We were not able to detect any binding for V22D with ITC methods. This study clearly demonstrates that the single mutation at position 22 of αII, a region critical to the function of nonerythroid α spectrin, may lead to a reduced level of spectrin tetramers and abnormal spectrin-based membrane skeleton. These abnormalities could cause abnormal neural activities in cells.
Asunto(s)
Calorimetría/métodos , Multimerización de Proteína , Espectrina/metabolismo , Técnicas del Sistema de Dos Híbridos , Recuento de Colonia Microbiana , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
Spectrin tetramerization is important for the erythrocyte to maintain its unique shape, elasticity and deformability. We used recombinant model proteins to show the importance of one residue (G46) in the erythroid alpha-spectrin junction region that affects spectrin tetramer formation. The G46 residue in the erythroid spectrin N-terminal junction region is the only residue that differs from that in non-erythroid spectrin. The corresponding residue is R37. We believe that this difference may be, at least in part, responsible for the 15-fold difference in the equilibrium constants of erythroid and non-erythroid tetramer formation. In this study, we replaced the Gly residue with Ala, Arg or Glu residues in an erythroid alpha-spectrin model protein to give G46A, G46R or G46E, respectively. We found that their association affinities with a beta-spectrin model protein were quite different from each other. G46R exhibited a 10-fold increase and G46E exhibited a 16-fold decrease, whereas G46A showed little difference, when compared with the wild type. The thermal and urea denaturation experiments showed insignificant structural change in G46R. Thus, the differences in affinity were due to differences in local, specific interactions, rather than conformational differences in these variants. An intra-helical salt bridge in G46R may stabilize the partial domain single helix in alpha-spectrin, Helix C', to allow a more stable helical bundling in the alphabeta complex in spectrin tetramers. These results not only showed the importance of residue G46 in erythroid alpha-spectrin, but also provided insights toward the differences in association affinity between erythroid and non-erythroid spectrin to form spectrin tetramers.
Asunto(s)
Eritrocitos/química , Glicina/química , Espectrina/química , Sustitución de Aminoácidos , Mutagénesis Sitio-Dirigida , Desnaturalización Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrina/genética , Espectrina/metabolismoRESUMEN
Calpains and caspases are ubiquitous cysteine proteases that are associated with a variety of cellular pathways. Calpains are involved in processes such as long term potentiation, cell motility and apoptosis, and have been shown to cleave non-erythroid (brain) alpha- and beta-spectrin and erythroid beta-spectrin. The cleavage of erythroid alpha-spectrin by calpain has not been reported. Caspases play an important role in the initiation and execution of apoptosis, and have been shown to cleave non-erythroid but not erythroid spectrin. We have studied the effect of spectrin fragments on calpain and caspase activities. The erythroid and non-erythroid spectrin fragments used were from the N-terminal region of alpha-spectrin, and C-terminal region of beta-spectrin, both consisting of regions involved in spectrin tetramer formation. We observed that the all spectrin fragments exhibited a concentration-dependent inhibitory effect on calpain, but not caspase activity. It is clear that additional studies are warranted to determine the physiological significance of calpain inhibition by spectrin fragments. Our findings suggest that calpain activity is modulated by the presence of spectrin partial domains at the tetramerization site. It is not clear whether the inhibitory effect is substrate specific or is a general effect. Further studies of this inhibitory effect may lead to the identification and development of new therapeutic agents specifically for calpains, but not for caspases. Proteins/peptides with a coiled coil helical conformation should be studied for potential inhibitory effects on calpain activity.
Asunto(s)
Calpaína/metabolismo , Caspasas/metabolismo , Espectrina/metabolismo , Apoptosis , Calpaína/antagonistas & inhibidores , Humanos , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrina/química , Espectrina/genéticaRESUMEN
This perspective provides an overview of the evolution of antibiotic discovery from a largely phenotypic-based effort, through an intensive structure-based design focus, to a more holistic approach today. The current focus on antibiotic development incorporates assay and discovery conditions that replicate the host environment as much as feasible. They also incorporate several strategies, including target identification and validation within the whole cell environment, a variety of target deconvolution methods, and continued refinement of structure-based design approaches.
Asunto(s)
Antibacterianos/farmacología , Descubrimiento de Drogas , Antibacterianos/química , Biopelículas , Diseño de Fármacos , Desarrollo de Medicamentos , Quimioterapia Combinada , Genómica , MetabolómicaRESUMEN
The wide use of the antimicrobial agent/biocide, triclosan, promotes triclosan-resistant bacterial strains, including Staphylococcus aureus, as well as leads to accumulation in the aquatic and terrestrial environments. Knowledge of the molecular actions of triclosan on S. aureus is needed to understand the consequence of triclosan resistance and environmental accumulation of triclosan on S. aureus resistant strains, as well as to develop biphenyl ether analogs as antibiotic candidates. Triclosan inhibits an essential enzyme in the fatty acid biosynthetic pathway, the reduced nicotinamide adenine dinucleotide (NADH)/reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent enoyl-acyl carrier protein (enoyl-ACP) reductase, or FabI. In this study, we used error-prone polymerase chain reaction (epPCR) to generate mutations in the S. aureus FabI enzyme. Instead of using an elaborate FabI enzyme activity assay that involves ACP-linked substrates to determine whether triclosan inhibits the enzyme activities of individual FabI mutants, we used an efficient and economical assay that we developed, based on thermal shift principles, to screen for triclosan binding to FabI mutants in cells. We identified four active-site mutations. More interestingly, we also identified nine triclosan-resistant mutations distant from the active site (G113V, Y123H, S166N, N220I, G227C, A230T, V241I, F252I, and H253P) but located in disparate positions in the monomer-monomer and dimer-dimer interface regions in S. aureus FabI. We suggest that these sites may serve as potential allosteric sites for designing potential therapeutic inhibitors that offer advantages in selectivity since allosteric sites are less evolutionarily conserved.
RESUMEN
Glutamate racemase (RacE) is a bacterial enzyme that converts l-glutamate to d-glutamate, an essential precursor for peptidoglycan synthesis. In prior work, we have shown that both isoforms cocrystallize with d-glutamate as dimers, and the enzyme is in a closed conformation with limited access to the active site [May, M., et al. (2007) J. Mol. Biol. 371, 1219-1237]. The active site of RacE2 is especially restricted. We utilize several computational and experimental approaches to understand the overall conformational dynamics involved during catalysis when the ligand enters and the product exits the active site. Our steered molecular dynamics simulations and normal-mode analysis results indicate that the monomeric form of the enzyme is more flexible than the native dimeric form. These results suggest that the monomeric enzyme might be more active than the dimeric form. We thus generated site-specific mutations that disrupt dimerization and find that the mutants exhibit significantly higher catalytic rates in the d-Glu to l-Glu reaction direction than the native enzyme. Low-resolution models restored from solution X-ray scattering studies correlate well with the first six normal modes of the dimeric form of the enzyme, obtained from NMA. Thus, along with the local active site residues, global domain motions appear to be implicated in the catalytically relevant structural dynamics of this enzyme and suggest that increased flexibility may accelerate catalysis. This is a novel observation that residues distant from the catalytic site restrain catalytic activity through formation of the dimer structure.
Asunto(s)
Isomerasas de Aminoácido/metabolismo , Isomerasas de Aminoácido/química , Isomerasas de Aminoácido/genética , Biocatálisis , Cromatografía en Gel , Dimerización , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Dispersión de RadiaciónRESUMEN
The functional roles of residues 21-43 and 55-59 in the alpha-spectrin N-terminal region in forming tetramers were determined by the introduction of mutations at each of these positions. We measured association affinities for tetramer formation (K(d)), which can be used to predict clinical severity, of these mutants. A total of nine residues critical for association with beta-spectrin were found. The mutations of six of these residues have already been known to cause hereditary elliptocytosis or hereditary pyropoikilocytosis. Clinical symptoms associated with three mutations of residues 23, 57 and 58 have not yet been reported. We suggest that these mutations may also introduce abnormalities to erythrocytes.
Asunto(s)
Eritrocitos/química , Mutación , Espectrina/genética , Espectroscopía de Resonancia por Spin del Electrón/métodos , Humanos , Fragmentos de Péptidos/genética , Unión Proteica , Relación Estructura-ActividadRESUMEN
We previously determined the solution structures of the first 156 residues of human erythroid alpha-spectrin (SpalphaI-1-156, or simply Spalpha). Spalpha consists of the tetramerization site of alpha-spectrin and associates with a model beta-spectrin protein (Spbeta) with an affinity similar to that of native alpha- and beta-spectrin. Upon alphabeta-complex formation, our previous results indicate that there is an increase in helicity in the complex, suggesting conformational change in either Spalpha or Spbeta or in both. We have now used isothermal titration calorimetry, circular dichroism, static and dynamic light scattering, and solution NMR methods to investigate properties of the complex as well as the conformation of Spalpha in the complex. The results reveal a highly asymmetric complex, with a Perrin shape parameter of 1.23, which could correspond to a prolate ellipsoid with a major axis of about five and a minor axis of about one. We identified 12 residues, five prior to and seven following the partial domain helix in Spalpha that moved freely relative to the structural domain in the absence of Spbeta but when in the complex moved with a mobility similar to that of the structural domain. Thus, it appears that the association with Spbeta induced an unstructured-to-helical conformational transition in these residues to produce a rigid and asymmetric complex. Our findings may provide insight toward understanding different association affinities of alphabeta-spectrin at the tetramerization site for erythroid and non-erythroid spectrin and a possible mechanism to understand some of the clinical mutations, such as L49F of alpha-spectrin, which occur outside the functional partial domain region.
Asunto(s)
Eritrocitos/metabolismo , Espectrina/química , Calorimetría/métodos , Cromatografía/métodos , Dicroismo Circular , Humanos , Espectroscopía de Resonancia Magnética/métodos , Modelos Estadísticos , Peso Molecular , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Dispersión de RadiaciónRESUMEN
The N-terminal region of non-erythroid alpha spectrin (Sp alpha II) is responsible for interacting with its binding partner, beta spectrin, to form functional spectrin tetramers. We used a yeast-two-hybrid system, with an N-terminal segment of alpha spectrin representing the functional tetramerization site, as a bait to screen human brain c-DNA library for proteins that interact with the alpha spectrin segment. In addition to several beta spectrin isoforms, we identified 14 proteins that interact with Sp alpha II. Seven of the 14 were matched to 6 known proteins: Duo protein, Lysyl-tRNA synthetase, TBP associated factor 1, two isoforms (b and c) of a protein kinase A interacting protein and Zinc finger protein 333 (2 different segments). Four of the 6 proteins are located primarily in the nucleus, suggesting that spectrin plays important roles in nuclear functions. The remaining 7 proteins were unknown to the protein data base. Structural predictions show that many of the 14 proteins consist of a large portion of unstructured regions, suggesting that many of these proteins fold into a rather flexible conformation. It is interesting to note that all but 3 of the 14 proteins are predicted to consist of one to four coiled coils (amphiphilic helices). A mutation in Sp alpha II, V22D, which interferes with the coiled coil bundling of Sp alpha II with beta spectrin, also affects Sp alpha II interaction with Duo protein, TBP associated factor 1 and Lysyl-tRNA synthetase, suggesting that they may compete with beta spectrin for interaction with Sp alpha II. Future structural and functional studies of these proteins to provide interaction mechanisms will no doubt lead to a better understanding of brain physiology and pathophysiology.