RESUMEN
Methylation of 2-deoxyuridine-5'-monophosphate (dUMP) at the C5 position by the obligate dimeric thymidylate synthase (TSase) in the sole de novo biosynthetic pathway to thymidine 5'-monophosphate (dTMP) proceeds by forming a covalent ternary complex with dUMP and cosubstrate 5,10-methylenetetrahydrofolate. The crystal structure of an analog of this intermediate gives important mechanistic insights but does not explain the half-of-the-sites activity of the enzyme. Recent experiments showed that the C5 proton and the catalytic Cys are eliminated in a concerted manner from the covalent ternary complex to produce a noncovalent bisubstrate intermediate. Here, we report the crystal structure of TSase with a close synthetic analog of this intermediate in which it has partially reacted with the enzyme but in only one protomer, consistent with the half-of-the-sites activity of this enzyme. Quantum mechanics/molecular mechanics simulations confirmed that the analog could undergo catalysis. The crystal structure shows a new water 2.9 Å from the critical C5 of the dUMP moiety, which in conjunction with other residues in the network, may be the elusive general base that abstracts the C5 proton of dUMP during the reaction.
Asunto(s)
Timidilato Sintasa/química , Dominio Catalítico , Cristalografía por Rayos X , Cinética , Modelos Moleculares , Timidilato Sintasa/metabolismoRESUMEN
The lactose permease of Escherichia coli (LacY), a dynamic polytopic membrane transport protein, catalyzes galactoside/H+ symport and operates by an alternating access mechanism that exhibits multiple conformations, the distribution of which is altered by sugar-binding. Camelid nanobodies were made against a double-mutant Gly46 â Trp/Gly262 â Trp (LacYWW) that produces an outward-open conformation, as opposed to the cytoplasmic open-state crystal structure of WT LacY. Nanobody 9047 (Nb9047) stabilizes WT LacY in a periplasmic-open conformation. Here, we describe the X-ray crystal structure of a complex between LacYWW, the high-affinity substrate analog 4-nitrophenyl-α-d-galactoside (NPG), and Nb9047 at 3-Å resolution. The present crystal structure demonstrates that Nb9047 binds to the periplasmic face of LacY, primarily to the C-terminal six-helical bundle, while a flexible loop of the Nb forms a bridge between the N- and C-terminal halves of LacY across the periplasmic vestibule. The bound Nb partially covers the vestibule, yet does not affect the on-rates or off-rates for the substrate binding to LacYWW, which implicates dynamic flexibility of the Nb-LacYWW complex. Nb9047-binding neither changes the overall structure of LacYWW with bound NPG, nor the positions of side chains comprising the galactoside-binding site. The current NPG-bound structure exhibits a more occluded periplasmic vestibule than seen in a previous structure of a (different Nb) apo-LacYWW/Nb9039 complex that we argue is caused by sugar-binding, with major differences located at the periplasmic ends of transmembrane helices in the N-terminal half of LacY.
Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas de Transporte de Monosacáridos/química , Anticuerpos de Dominio Único/química , Simportadores/química , Cristalografía por Rayos X , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Transporte de Monosacáridos/genética , Mutación , Estructura Cuaternaria de Proteína , Simportadores/genéticaRESUMEN
In Escherichia coli thymidylate synthase (EcTS), rate-determining hydride transfer from the cofactor 5,10-methylene-5,6,7,8-tetrahydrofolate to the intermediate 5-methylene-2'-deoxyuridine 5'-monophosphate occurs by hydrogen tunneling, requiring precise alignment of reactants and a closed binding cavity, sealed by the C-terminal carboxyl group. Mutations that destabilize the closed conformation of the binding cavity allow small molecules such as ß-mercaptoethanol (ß-ME) to enter the active site and compete with hydride for addition to the 5-methylene group of the intermediate. The C-terminal deletion mutant of EcTS produced the ß-ME adduct in proportions that varied dramatically with cofactor concentration, from 50% at low cofactor concentrations to 0% at saturating cofactor conditions, suggesting communication between active sites. We report the 2.4 Å X-ray structure of the C-terminal deletion mutant of E. coli TS in complex with a substrate and a cofactor analogue, CB3717. The structure is asymmetric, with reactants aligned in a manner consistent with hydride transfer in only one active site. In the second site, CB3717 has shifted to a site where the normal cofactor would be unlikely to form 5-methylene-2'-deoxyuridine 5'-monophosphate, consistent with no formation of the ß-ME adduct. The structure shows how the binding of the cofactor at one site triggers hydride transfer and borrows needed stabilization from substrate binding at the second site. It indicates pathways through the dimer interface that contribute to allostery relevant to half-sites reactivity.
Asunto(s)
Escherichia coli/química , Ácido Fólico/análogos & derivados , Conformación Proteica , Quinazolinas/química , Timidilato Sintasa/química , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Escherichia coli/enzimología , Ácido Fólico/química , Hidrógeno/química , Modelos Moleculares , Mutación , Especificidad por Sustrato , Tetrahidrofolatos/química , Timidilato Sintasa/genéticaRESUMEN
Cancerous cells have an acutely increased demand for energy, leading to increased levels of human glucose transporter 1 (hGLUT1). This up-regulation suggests hGLUT1 as a target for therapeutic inhibitors addressing a multitude of cancer types. Here, we present three inhibitor-bound, inward-open structures of WT-hGLUT1 crystallized with three different inhibitors: cytochalasin B, a nine-membered bicyclic ring fused to a 14-membered macrocycle, which has been described extensively in the literature of hGLUTs, and two previously undescribed Phe amide-derived inhibitors. Despite very different chemical backbones, all three compounds bind in the central cavity of the inward-open state of hGLUT1, and all binding sites overlap the glucose-binding site. The inhibitory action of the compounds was determined for hGLUT family members, hGLUT1-4, using cell-based assays, and compared with homology models for these hGLUT members. This comparison uncovered a probable basis for the observed differences in inhibition between family members. We pinpoint regions of the hGLUT proteins that can be targeted to achieve isoform selectivity, and show that these same regions are used for inhibitors with very distinct structural backbones. The inhibitor cocomplex structures of hGLUT1 provide an important structural insight for the design of more selective inhibitors for hGLUTs and hGLUT1 in particular.
Asunto(s)
Citocalasinas/química , Transportador de Glucosa de Tipo 1/antagonistas & inhibidores , Transportador de Glucosa de Tipo 1/ultraestructura , Glucosa/química , Fenilalanina/análogos & derivados , Secuencia de Aminoácidos , Sitios de Unión , Simulación por Computador , Secuencia Conservada , Humanos , Modelos Químicos , Modelos Moleculares , Fenilalanina/química , Unión Proteica , Conformación ProteicaRESUMEN
OBJECTIVE: Early initiation of antiretroviral treatment (ART) at CD4 cell count ≥ 500 cells per microliter reduces morbidity and mortality in HIV-infected adults. We determined the proportion of HIV-infected people with high viral load (VL) for whom transmission prevention would be an additional benefit of early treatment. DESIGN: A randomly selected subset of a nationally representative sample of HIV-infected adults in Swaziland in 2012. METHODS: Eight to 12 months after a national survey to determine adult HIV prevalence, 1067 of 5802 individuals identified as HIV-infected were asked to participate in a follow-up cross-sectional assessment. CD4 cell enumeration, VL measurements, and ART status were obtained to estimate the proportion of currently untreated adults and of the entire HIV-infected population with high VL (≥ 1000 copies/mL) whose treatment under a test-and-treat or VL threshold eligibility strategy would reduce HIV transmission. RESULTS: Of the 927 (87% of 1067) participants enrolled, 466 (50%) reported no ART use. Among them, 424 (91%) had VL ≥ 1000 copies per milliliter; of these, 148 (35%) were eligible for ART at the then existing CD4 count threshold of <350 cells per microliter; an additional 107 (25%) were eligible with expanded CD4 criterion of <500 cells per microliter; and 169 (40%) remained ART ineligible. Thus, 36% of the 466 currently untreated and 18% of the total 927 had high VL yet remained ART ineligible under a CD4 criterion of <500 cells per microliter. CONCLUSIONS: A test-and-treat or VL threshold for treatment eligibility is necessary to maximize the HIV transmission prevention benefits of ART.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/epidemiología , Adolescente , Adulto , Recuento de Linfocito CD4 , Estudios Transversales , Esuatini/epidemiología , Femenino , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Cooperación del Paciente , Guías de Práctica Clínica como Asunto , Prevalencia , Carga Viral , Adulto JovenRESUMEN
The X-ray crystal structure of a conformationally constrained mutant of the Escherichia coli lactose permease (the LacY double-Trp mutant Gly-46âTrp/Gly-262âTrp) with bound p-nitrophenyl-α-d-galactopyranoside (α-NPG), a high-affinity lactose analog, is described. With the exception of Glu-126 (helix IV), side chains Trp-151 (helix V), Glu-269 (helix VIII), Arg-144 (helix V), His-322 (helix X), and Asn-272 (helix VIII) interact directly with the galactopyranosyl ring of α-NPG to provide specificity, as indicated by biochemical studies and shown directly by X-ray crystallography. In contrast, Phe-20, Met-23, and Phe-27 (helix I) are within van der Waals distance of the benzyl moiety of the analog and thereby increase binding affinity nonspecifically. Thus, the specificity of LacY for sugar is determined solely by side-chain interactions with the galactopyranosyl ring, whereas affinity is increased by nonspecific hydrophobic interactions with the anomeric substituent.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Proteínas de Transporte de Monosacáridos/química , Proteínas de Transporte de Monosacáridos/metabolismo , Nitrofenilgalactósidos/metabolismo , Protones , Simportadores/química , Simportadores/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Ligandos , Modelos Moleculares , Estructura Secundaria de Proteína , Electricidad Estática , Especificidad por SustratoRESUMEN
WOPR-domain proteins are found throughout the fungal kingdom where they function as master regulators of cell morphology and pathogenesis. Genetic and biochemical experiments previously demonstrated that these proteins bind to specific DNA sequences and thereby regulate transcription. However, their primary sequence showed no relationship to any known DNA-binding domain, and the basis for their ability to recognize DNA sequences remained unknown. Here, we describe the 2.6-Å crystal structure of a WOPR domain in complex with its preferred DNA sequence. The structure reveals that two highly conserved regions, separated by an unconserved linker, form an interdigitated ß-sheet that is tilted into the major groove of DNA. Although the main interaction surface is in the major groove, the highest-affinity interactions occur in the minor groove, primarily through a deeply penetrating arginine residue. The structure reveals a new, unanticipated mechanism by which proteins can recognize specific sequences of DNA.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Hongos/patogenicidad , Aminoácidos/metabolismo , Secuencia de Bases , Secuencia Conservada/genética , Cristalografía por Rayos X , ADN de Hongos/química , ADN de Hongos/metabolismo , Evolución Molecular , Hongos/metabolismo , Regulación Fúngica de la Expresión Génica , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Filogenia , Unión Proteica , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Relación Estructura-Actividad , Transactivadores/química , Transactivadores/metabolismo , Activación Transcripcional/genéticaRESUMEN
Bitopic integral membrane proteins with a single transmembrane helix play diverse roles in catalysis, cell signaling, and morphogenesis. Complete monospanning protein structures are needed to show how interaction between the transmembrane helix and catalytic domain might influence association with the membrane and function. We report crystal structures of full-length Saccharomyces cerevisiae lanosterol 14α-demethylase, a membrane monospanning cytochrome P450 of the CYP51 family that catalyzes the first postcyclization step in ergosterol biosynthesis and is inhibited by triazole drugs. The structures reveal a well-ordered N-terminal amphipathic helix preceding a putative transmembrane helix that would constrain the catalytic domain orientation to lie partly in the lipid bilayer. The structures locate the substrate lanosterol, identify putative substrate and product channels, and reveal constrained interactions with triazole antifungal drugs that are important for drug design and understanding drug resistance.
Asunto(s)
Dominio Catalítico/genética , Sistema Enzimático del Citocromo P-450/química , Membrana Dobles de Lípidos/metabolismo , Modelos Moleculares , Conformación Proteica , Proteínas de Saccharomyces cerevisiae/química , Cromatografía de Afinidad , Cromatografía en Gel , CristalizaciónRESUMEN
Here we describe the X-ray crystal structure of a double-Trp mutant (Gly46âTrp/Gly262âTrp) of the lactose permease of Escherichia coli (LacY) with a bound, high-affinity lactose analog. Although thought to be arrested in an open-outward conformation, the structure is almost occluded and is partially open to the periplasmic side; the cytoplasmic side is tightly sealed. Surprisingly, the opening on the periplasmic side is sufficiently narrow that sugar cannot get in or out of the binding site. Clearly defined density for a bound sugar is observed at the apex of the almost occluded cavity in the middle of the protein, and the side chains shown to ligate the galactopyranoside strongly confirm more than two decades of biochemical and spectroscopic findings. Comparison of the current structure with a previous structure of LacY with a covalently bound inactivator suggests that the galactopyranoside must be fully ligated to induce an occluded conformation. We conclude that protonated LacY binds D-galactopyranosides specifically, inducing an occluded state that can open to either side of the membrane.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Escherichia coli/enzimología , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Aminoácidos/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Isopropil Tiogalactósido/química , Isopropil Tiogalactósido/metabolismo , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Electricidad Estática , Especificidad por SustratoRESUMEN
N-[4-[2-Propyn-1-yl[(6S)-4,6,7,8-tetrahydro-2-(hydroxymethyl)-4-oxo-3H-cyclopenta[g]quinazolin-6-yl]amino]benzoyl]-l-γ-glutamyl-d-glutamic acid 1 (BGC 945, now known as ONX 0801), is a small molecule thymidylate synthase (TS) inhibitor discovered at the Institute of Cancer Research in London. It is licensed by Onyx Pharmaceuticals and is in phase 1 clinical studies. It is a novel antifolate drug resembling TS inhibitors plevitrexed and raltitrexed that combines enzymatic inhibition of thymidylate synthase with α-folate receptor-mediated targeting of tumor cells. Thus, it has potential for efficacy with lower toxicity due to selective intracellular accumulation through α-folate receptor (α-FR) transport. The α-FR, a cell-surface receptor glycoprotein, which is overexpressed mainly in ovarian and lung cancer tumors, has an affinity for 1 similar to that for its natural ligand, folic acid. This study describes a novel synthesis of 1, an X-ray crystal structure of its complex with Escherichia coli TS and 2'-deoxyuridine-5'-monophosphate, and a model for a similar complex with human TS.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Antagonistas del Ácido Fólico/farmacología , Quinazolinas/farmacología , Timidilato Sintasa/antagonistas & inhibidores , Cristalografía por Rayos X , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Escherichia coli/enzimología , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Antagonistas del Ácido Fólico/síntesis química , Antagonistas del Ácido Fólico/metabolismo , Humanos , Modelos Químicos , Modelos Moleculares , Estructura Molecular , Neoplasias/enzimología , Neoplasias/patología , Unión Proteica , Estructura Terciaria de Proteína , Quinazolinas/síntesis química , Quinazolinas/metabolismo , Timidilato Sintasa/química , Timidilato Sintasa/metabolismoRESUMEN
Thymidylate synthase (TSase) produces the sole intracellular de novo source of thymidine (i.e., the DNA base T) and thus is a common target for antibiotic and anticancer drugs. Mg(2+) has been reported to affect TSase activity, but the mechanism of this interaction has not been investigated. Here we show that Mg(2+) binds to the surface of Escherichia coli TSase and affects the kinetics of hydride transfer at the interior active site (16 Å away). Examination of the crystal structures identifies a Mg(2+) near the glutamyl moiety of the folate cofactor, providing the first structural evidence for Mg(2+) binding to TSase. The kinetics and NMR relaxation experiments suggest that the weak binding of Mg(2+) to the protein surface stabilizes the closed conformation of the ternary enzyme complex and reduces the entropy of activation on the hydride transfer step. Mg(2+) accelerates the hydride transfer by ~7-fold but does not affect the magnitude or temperature dependence of the intrinsic kinetic isotope effect. These results suggest that Mg(2+) facilitates the protein motions that bring the hydride donor and acceptor together, but it does not change the tunneling ready state of the hydride transfer. These findings highlight how variations in cellular Mg(2+) concentration can modulate enzyme activity through long-range interactions in the protein, rather than binding at the active site. The interaction of Mg(2+) with the glutamyl tail of the folate cofactor and nonconserved residues of bacterial TSase may assist in designing antifolates with polyglutamyl substitutes as species-specific antibiotic drugs.
Asunto(s)
Magnesio/química , Timidilato Sintasa/química , Sitios de Unión , Escherichia coli/enzimología , Magnesio/metabolismo , Modelos Moleculares , Estructura Molecular , Propiedades de Superficie , Timidilato Sintasa/metabolismo , Agua/química , Agua/metabolismoRESUMEN
Enzymes in lipid metabolism acquire and deliver hydrophobic substrates and products from within lipid bilayers. The structure at 2.55 Å of one isozyme of a constitutive enzyme in lipid A biosynthesis, LpxI from Caulobacter crescentus, has a novel fold. Two domains close around a completely sequestered substrate, UDP-2,3-diacylglucosamine, and open to release products either to the neighboring enzyme in a putative multienzyme complex or to the bilayer. Mutation analysis identifies Asp225 as key to Mg(2+)-catalyzed diphosphate hydrolysis. These structures provide snapshots of the enzymatic synthesis of a critical lipid A precursor.
Asunto(s)
Caulobacter crescentus/enzimología , Lípido A/biosíntesis , Modelos Moleculares , Conformación Proteica , Pirofosfatasas/química , Secuencia de Aminoácidos , Cristalización , Análisis Mutacional de ADN , Glucolípidos/metabolismo , Isoenzimas/química , Espectrometría de Masas , Datos de Secuencia Molecular , Estructura Molecular , Pliegue de Proteína , Pirofosfatasas/genética , UltracentrifugaciónRESUMEN
The role of protein flexibility in enzyme-catalyzed activation of chemical bonds is an evolving perspective in enzymology. Here we examine the role of protein motions in the hydride transfer reaction catalyzed by thymidylate synthase (TSase). Being remote from the chemical reaction site, the Y209W mutation of Escherichia coli TSase significantly reduces the protein activity, despite the remarkable similarity between the crystal structures of the wild-type and mutant enzymes with ligands representing their Michaelis complexes. The most conspicuous difference between these two crystal structures is in the anisotropic B-factors, which indicate disruption of the correlated atomic vibrations of protein residues in the mutant. This dynamically altered mutant allows a variety of small thiols to compete for the reaction intermediate that precedes the hydride transfer, indicating disruption of motions that preorganize the protein environment for this chemical step. Although the mutation causes higher enthalpy of activation of the hydride transfer, it only shows a small effect on the temperature dependence of the intrinsic KIE, suggesting marginal changes in the geometry and dynamics of the H-donor and -acceptor at the tunneling ready state. These observations suggest that the mutation disrupts the concerted motions that bring the H-donor and -acceptor together during the pre- and re-organization of the protein environment. The integrated structural and kinetic data allow us to probe the impact of protein motions on different time scales of the hydride transfer reaction within a complex enzymatic mechanism.
Asunto(s)
Escherichia coli/enzimología , Timidilato Sintasa/metabolismo , Biocatálisis , Cristalografía por Rayos X , Cinética , Modelos Moleculares , Estructura Molecular , Mutación , Timidilato Sintasa/química , Timidilato Sintasa/genéticaRESUMEN
EspR is a transcriptional regulator that activates the ESX-1 secretion system during Mycobacterium tuberculosis infection and is critical for pathogenesis. It is unique among DNA-binding proteins as it is secreted as part of a feedback regulatory loop that serves to mitigate transcriptional activity. Here we report the crystal structure of a functional EspR dimer at 2.5-Å resolution. The amino-terminal half of EspR is a helix-turn-helix (HTH) DNA-binding domain and the carboxy terminus consists of a dimerization domain with similarity to the SinR:SinI sporulation regulator of Bacillus subtilis. Surprisingly, the HTH domains of EspR are arranged in an unusual conformation in which they are splayed at an oblique angle to each other, suggesting that EspR binds DNA in a profoundly different way than most other known HTH regulators. By mapping the EspR binding sites in the espACD promoter, using both in vivo and in vitro binding assays, we show that the EspR operators are located unusually far from the promoter. The EspR dimer binds to these sites cooperatively, but the two "half-sites" contacted by each DNA recognition motif are separated by 177 base pairs. The distinctive structure of EspR and the exceptional arrangement of its operator contacts suggest that it could promote DNA looping in its target promoter. We hypothesize that direct DNA looping mediated by single-site binding of each EspR monomer may facilitate transcriptional control of this important virulence system.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de Unión al ADN/química , ADN/química , Secuencias Hélice-Giro-Hélice , Mycobacterium tuberculosis/química , Estructura Cuaternaria de Proteína , Factores de Transcripción/química , Sitios de Unión , Cristalografía por Rayos X , ADN/metabolismo , Modelos Moleculares , Mycobacterium tuberculosis/patogenicidad , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , Multimerización de Proteína , VirulenciaRESUMEN
Escherichia coli pseudouridine synthase RluF is dedicated to modifying U2604 in a stem-loop of 23S RNA, while a homologue, RluB, modifies the adjacent base, U2605. Both uridines are in the same RNA stem, separated by approximately 4 A. The 3.0 A X-ray crystal structure of RluF bound to the isolated stem-loop, in which U2604 is substituted by 5-fluorouridine to prevent catalytic turnover, shows RluF distinguishes closely spaced bases in similar environments by a selectivity mechanism based on a frameshift in base pairing. The RNA stem-loop is bound to a conserved binding groove in the catalytic domain. A base from a bulge in the stem, A2602, has folded into the stem, forcing one strand of the RNA stem to translate by one position and thus positioning U2604 to flip into the active site. RluF does not modify U2604 in mutant stem-loops that lack the A2602 bulge and shows dramatically higher activity for a stem-loop with a mutation designed to facilitate A2602 refolding into the stem with concomitant RNA strand translation. Residues whose side chains contact rearranged bases in the bound stem-loop, while conserved among RluFs, are not conserved between RluFs and RluBs, suggesting that RluB does not bind to the rearranged stem loop.
Asunto(s)
Emparejamiento Base , Proteínas de Escherichia coli , Hidroliasas , Conformación de Ácido Nucleico , Conformación Proteica , ARN , Ribosomas/genética , Dominio Catalítico , Cristalización , Cristalografía por Rayos X , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Hidroliasas/química , Hidroliasas/genética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , ARN/química , ARN/genéticaRESUMEN
The enzyme thymidylate synthase (TS) catalyzes the reductive methylation of 2'-deoxyuridine 5'-monophosphate (dUMP) to 2'-deoxythymidine 5'-monophosphate. Using kinetic and X-ray crystallography experiments, we have examined the role of the highly conserved Tyr-261 in the catalytic mechanism of TS. While Tyr-261 is distant from the site of methyl transfer, mutants at this position show a marked decrease in enzymatic activity. Given that Tyr-261 forms a hydrogen bond with the dUMP 3'-O, we hypothesized that this interaction would be important for substrate binding, orientation, and specificity. Our results, surprisingly, show that Tyr-261 contributes little to these features of the mechanism of TS. However, the residue is part of the structural core of closed ternary complexes of TS, and conservation of the size and shape of the Tyr side chain is essential for maintaining wild-type values of kcat/Km. Moderate increases in Km values for both the substrate and cofactor upon mutation of Tyr-261 arise mainly from destabilization of the active conformation of a loop containing a dUMP-binding arginine. Besides binding dUMP, this loop has a key role in stabilizing the closed conformation of the enzyme and in shielding the active site from the bulk solvent during catalysis. Changes to atomic vibrations in crystals of a ternary complex of Escherichia coli Tyr261Trp are associated with a greater than 2000-fold drop in kcat/Km. These results underline the important contribution of dynamics to catalysis in TS.
Asunto(s)
Nucleótidos de Desoxiuracil/metabolismo , Timidilato Sintasa/metabolismo , Tirosina/metabolismo , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Nucleótidos de Desoxiuracil/química , Escherichia coli/química , Escherichia coli/enzimología , Escherichia coli/genética , Enlace de Hidrógeno , Cinética , Modelos Moleculares , Estructura Molecular , Mutación , Unión Proteica , Conformación Proteica , Especificidad por Sustrato , Timidilato Sintasa/química , Timidilato Sintasa/genética , Tirosina/química , Tirosina/genéticaRESUMEN
Unfolded proteins in the endoplasmic reticulum (ER) activate the ER transmembrane sensor Ire1 to trigger the unfolded protein response (UPR), a homeostatic signaling pathway that adjusts ER protein folding capacity according to need. Ire1 is a bifunctional enzyme, containing cytoplasmic kinase and RNase domains whose roles in signal transduction downstream of Ire1 are understood in some detail. By contrast, the question of how its ER-luminal domain (LD) senses unfolded proteins has remained an enigma. The 3.0-A crystal structure and consequent structure-guided functional analyses of the conserved core region of the LD (cLD) leads us to a proposal for the mechanism of response. cLD exhibits a unique protein fold and is sufficient to control Ire1 activation by unfolded proteins. Dimerization of cLD monomers across a large interface creates a shared central groove formed by alpha-helices that are situated on a beta-sheet floor. This groove is reminiscent of the peptide binding domains of major histocompatibility complexes (MHCs) in its gross architecture. Conserved amino acid side chains in Ire1 that face into the groove are shown to be important for UPR activation in that their mutation reduces the response. Mutational analyses suggest that further interaction between cLD dimers is required to form higher-order oligomers necessary for UPR activation. We propose that cLD directly binds unfolded proteins, which changes the quaternary association of the monomers in the membrane plane. The changes in the ER lumen in turn position Ire1 kinase domains in the cytoplasm optimally for autophosphorylation to initiate the UPR.
Asunto(s)
Retículo Endoplásmico/metabolismo , Secuencia de Aminoácidos , Animales , Northern Blotting , Western Blotting , Cristalografía por Rayos X , Citoplasma/metabolismo , Análisis Mutacional de ADN , Dimerización , Glutatión Transferasa/metabolismo , Humanos , Cinética , Luz , Complejo Mayor de Histocompatibilidad , Espectrometría de Masas , Glicoproteínas de Membrana/química , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Fosforilación , Filogenia , Plásmidos/metabolismo , Unión Proteica , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Proteínas Serina-Treonina Quinasas/química , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Ribonucleasas/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Dispersión de Radiación , Homología de Secuencia de Aminoácido , Transducción de Señal , Ultracentrifugación , Difracción de Rayos XRESUMEN
The ternary complex crystal structures of Cryptococcus neoformans and Escherichia coli thymidylate synthase (TS) suggest mechanisms of species-specific inhibition of a highly conserved protein. The 2.1 Angstrom structure of C. neoformans TS cocrystallized with substrate and the cofactor analog CB3717 shows that the binding sites for substrate and cofactor are highly conserved with respect to human TS, but that the structure of the cofactor-binding site of C. neoformans TS is less constrained by surrounding residues. This feature might allow C. neoformans TS to form TS-dUMP-inhibitor complexes with a greater range of antifolates than human TS. 3',3''-Dibromophenol-4-chloro-1,8-naphthalein (GA9) selectively inhibits both E. coli TS and C. neoformans TS (K(i) = 4 microM) over human TS (K(i) >> 245 microM). The E. coli TS-dUMP-GA9 complex is in an open conformation, similar to that of the apoenzyme crystal structure. The GA9-binding site overlaps the binding site of the pABA-glutamyl moiety of the cofactor. The fact that human apoTS can adopt an unusual fold in which the GA9-binding site is disordered may explain the poor affinity of GA9 for the human enzyme. These observations highlight the critical need to incorporate multiple target conformations in any computational attempt to facilitate drug discovery.
Asunto(s)
Cryptococcus neoformans/enzimología , Timidilato Sintasa/química , Algoritmos , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Diseño de Fármacos , Escherichia coli/metabolismo , Eliminación de Gen , Humanos , Cinética , Ligandos , Modelos Químicos , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Programas Informáticos , Especificidad de la Especie , TemperaturaRESUMEN
One of the fundamental aspects of HIV counselling for women is condom negotiation strategy development. The present research sought to identify condom request strategies used by Zimbabwean women and to determine which were most effective in persuading male partners to use condoms. Of six types of strategies used by women after a prevention intervention, one was significantly associated with consistent condom use 2 months later. Implications for the development of counselling and testing protocols are discussed.