RESUMEN
TANK-binding kinase 1 (TBK1) is a multifunctional kinase with an essential role in mitophagy, the selective clearance of damaged mitochondria. More than 90 distinct mutations in TBK1 are linked to amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia, including missense mutations that disrupt the abilities of TBK1 to dimerize, associate with the mitophagy receptor optineurin (OPTN), autoactivate, or catalyze phosphorylation. We investigated how ALS-associated mutations in TBK1 affect Parkin-dependent mitophagy using imaging to dissect the molecular mechanisms involved in clearing damaged mitochondria. Some mutations cause severe dysregulation of the pathway, while others induce limited disruption. Mutations that abolish either TBK1 dimerization or kinase activity were insufficient to fully inhibit mitophagy, while mutations that reduced both dimerization and kinase activity were more disruptive. Ultimately, both TBK1 recruitment and OPTN phosphorylation at S177 are necessary for engulfment of damaged mitochondra by autophagosomal membranes. Surprisingly, we find that ULK1 activity contributes to the phosphorylation of OPTN in the presence of either wild-type or kinase-inactive TBK1. In primary neurons, TBK1 mutants induce mitochondrial stress under basal conditions; network stress is exacerbated with further mitochondrial insult. Our study further refines the model for TBK1 function in mitophagy, demonstrating that some ALS-linked mutations likely contribute to disease pathogenesis by inducing mitochondrial stress or inhibiting mitophagic flux. Other TBK1 mutations exhibited much less impact on mitophagy in our assays, suggesting that cell-type-specific effects, cumulative damage, or alternative TBK1-dependent pathways such as innate immunity and inflammation also factor into the development of ALS in affected individuals.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Demencia Frontotemporal/genética , Mitofagia/genética , Mutación Missense/genética , Proteínas Serina-Treonina Quinasas/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Proteínas de Ciclo Celular/metabolismo , Predisposición Genética a la Enfermedad , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Cinética , Proteínas de Transporte de Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/genética , Mitocondrias/patología , Proteínas Mutantes/metabolismo , Estrés Oxidativo , Fosforilación , Dominios Proteicos , Multimerización de Proteína , Proteínas Serina-Treonina Quinasas/químicaRESUMEN
The recent use of organophosphate nerve agents in Syria, Malaysia, Russia, and the United Kingdom has reinforced the potential threat of their intentional release. These agents act through their ability to inhibit human acetylcholinesterase (hAChE; E.C. 3.1.1.7), an enzyme vital for survival. The toxicity of hAChE inhibition via G-series nerve agents has been demonstrated to vary widely depending on the G-agent used. To gain insight into this issue, the structures of hAChE inhibited by tabun, sarin, cyclosarin, soman, and GP were obtained along with the inhibition kinetics for these agents. Through this information, the role of hAChE active site plasticity in agent selectivity is revealed. With reports indicating that the efficacy of reactivators can vary based on the nerve agent inhibiting hAChE, human recombinatorially expressed hAChE was utilized to define these variations for HI-6 among various G-agents. To identify the structural underpinnings of this phenomenon, the structures of tabun, sarin, and soman-inhibited hAChE in complex with HI-6 were determined. This revealed how the presence of G-agent adducts impacts reactivator access and placement within the active site. These insights will contribute toward a path of next-generation reactivators and an improved understanding of the innate issues with the current reactivators.
Asunto(s)
Acetilcolinesterasa/metabolismo , Inhibidores de la Colinesterasa/efectos adversos , Agentes Nerviosos/efectos adversos , Oximas/efectos adversos , Compuestos de Piridinio/efectos adversos , Acetilcolinesterasa/química , Acetilcolinesterasa/aislamiento & purificación , Inhibidores de la Colinesterasa/química , Humanos , Estructura Molecular , Agentes Nerviosos/química , Oximas/química , Compuestos de Piridinio/químicaRESUMEN
Cation-chloride cotransporters (CCCs) regulate the movement of chloride across membranes, controlling physiological processes from cell volume maintenance to neuronal signaling. Human CCCs are clinical targets for existing diuretics and potentially additional indications. Here, we report the X-ray crystal structure of the soluble C-terminal regulatory domain of a eukaryotic potassium-chloride cotransporter, Caenorhabditis elegans KCC-1. We observe a core α/ß fold conserved among CCCs. Using structure-based sequence alignment, we analyze similarities and differences to the C-terminal domains of other CCC family members. We find that important regulatory motifs are in less-structured regions and residues important for dimerization are not widely conserved, suggesting that oligomerization and its effects may vary within the larger family. This snapshot of a eukaryotic KCC is a valuable starting point for the rational design of studies of cellular chloride regulation.
Asunto(s)
Proteínas de Caenorhabditis elegans/química , Simportadores/química , Animales , Proteínas de Caenorhabditis elegans/metabolismo , Cristalografía por Rayos X , Citosol/metabolismo , Células Eucariotas/metabolismo , Modelos Moleculares , Fosforilación , Dominios Proteicos , Multimerización de Proteína , Soluciones , Simportadores/metabolismo , Cotransportadores de K ClRESUMEN
Exonic DNA sequence variants in the Tbk1 gene associate with both sporadic and familial amyotrophic lateral sclerosis (ALS). Here, we examine functional defects in 25 missense TBK1 mutations, focusing on kinase activity and protein-protein interactions. We identified kinase domain (KD) mutations that abolish kinase activity or display substrate-specific defects in specific pathways, such as innate immunity and autophagy. By contrast, mutations in the scaffold dimerization domain (SDD) of TBK1 can cause the loss of kinase activity due to structural disruption, despite an intact KD. Familial ALS mutations in ubiquitin-like domain (ULD) or SDD display defects in dimerization; however, a subset retains kinase activity. These observations indicate that TBK1 dimerization is not required for kinase activation. Rather, dimerization seems to increase protein stability and enables efficient kinase-substrate interactions. Our study revealed many aspects of TBK1 activities affected by ALS mutations, highlighting the complexity of disease pathogenicity and providing insights into TBK1 activation mechanism.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Mutación Missense , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Esclerosis Amiotrófica Lateral/enzimología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Activación Enzimática , Humanos , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Fosforilación , Dominios Proteicos , Dominios y Motivos de Interacción de Proteínas/genética , Multimerización de Proteína , Proteínas Serina-Treonina Quinasas/química , Estabilidad Proteica , Serina/metabolismo , Especificidad por SustratoRESUMEN
Carbamates are esters of substituted carbamic acids that react with acetylcholinesterase (AChE) by initially transferring the carbamoyl group to a serine residue in the enzyme active site accompanied by loss of the carbamate leaving group followed by hydrolysis of the carbamoyl enzyme. This hydrolysis, or decarbamoylation, is relatively slow, and half-lives of carbamoylated AChEs range from 4â¯min to more than 30 days. Therefore, carbamates are effective AChE inhibitors that have been developed as insecticides and as therapeutic agents. In this report, we review recent data showing that decarbamoylation rate constants are independent of the ester leaving group for a series of carbamic acid esters with the same carbamoyl group and that decarbamoylation rate constants decreased by 800-fold when the alkyl substituents on the carbamoyl group increased in size from N-monomethyl- to N,N-diethyl-. We also review data showing that solvent deuterium oxide isotope effects for decarbamoylation decreased from 2.8 for N-monomethylcarbamoyl AChE to 1.1 for N,N-diethylcarbamoyl AChE, indicating a shift in the rate-limiting step from general acid-base catalysis to a likely conformational change in the distorted active site in N,N-diethylcarbamoyl AChE. The nature of such a conformational change is suggested from X-ray crystal structures of AChE phosphorylated by paraoxon.
Asunto(s)
Acetilcolinesterasa/metabolismo , Carbamatos/metabolismo , Acetilcolinesterasa/química , Carbamatos/química , Dominio Catalítico , Cristalografía por Rayos X , Cinética , Paraoxon/química , Paraoxon/metabolismoRESUMEN
Serving a critical role in neurotransmission, human acetylcholinesterase (hAChE) is the target of organophosphate nerve agents. Hence, there is an active interest in studying the mechanism of inhibition and recovery of enzymatic activity, which could lead to better countermeasures against nerve agents. As hAChE is found in different oligomeric assemblies, certain approaches to studying it have been problematic. Herein, we examine the biochemical and structural impact of monomerizing hAChE by using two mutations: L380R/F535K. The activities of monomeric hAChE L380R/F535K and dimeric hAChE were determined to be comparable utilizing a modified Ellman's assay. To investigate the influence of subunit-subunit interactions on the structure of hAChE, a 2.1 Å X-ray crystallographic structure was determined. Apart from minor shifts along the dimer interface, the overall structure of the hAChE L380R/F535K mutant is similar to that of dimeric hAChE. To probe whether the plasticity of the active site was overtly impacted by monomerizing hAChE, the kinetic constants of (PR/S ) - VX (ethyl({2-[bis(propan-2-yl)amino]ethyl}sulfanyl)(methyl)phosphinate) inhibition and subsequent rescue of hAChE L380R/F535K activity with HI-6 (1-(2'-hydroxyiminomethyl-1'-pyridinium)-3-(4'-carbamoyl-1-pyridinium)) were determined and found to be comparable to those of dimeric hAChE. Thus, hAChE L380R/F535K could be used as a substitute for dimeric hAChE when experimentally probing the ability of the hAChE active site to accommodate future nerve agent threats or judge the ability of new therapeutics to access the active site.
Asunto(s)
Acetilcolinesterasa/química , Acetilcolinesterasa/metabolismo , Acetilcolinesterasa/genética , Sitios de Unión , Humanos , Modelos Moleculares , Mutación , Conformación ProteicaRESUMEN
Over 50 years ago, the toxicity of irreversible organophosphate inhibitors targeting human acetylcholinesterase (hAChE) was observed to be stereospecific. The therapeutic reversal of hAChE inhibition by reactivators has also been shown to depend on the stereochemistry of the inhibitor. To gain clarity on the mechanism of stereospecific inhibition, the X-ray crystallographic structures of hAChE inhibited by a racemic mixture of VX (P R/S) and its enantiomers were obtained. Beyond identifying hAChE structural features that lend themselves to stereospecific inhibition, structures of the reactivator HI-6 bound to hAChE inhibited by VX enantiomers of varying toxicity, or in its uninhibited state, were obtained. Comparison of hAChE in these pre-reactivation and post-reactivation states along with enzymatic data reveals the potential influence of unproductive reactivator poses on the efficacy of these types of therapeutics. The recognition of structural features related to hAChE's stereospecificity toward VX shed light on the molecular influences of toxicity and their effect on reactivators. In addition to providing a better understanding of the innate issues with current reactivators, an avenue for improvement of reactivators is envisioned.
Asunto(s)
Acetilcolinesterasa/química , Reactivadores de la Colinesterasa/química , Compuestos Organotiofosforados/química , Oximas/química , Compuestos de Piridinio/química , Acetilcolinesterasa/genética , Acetilcolinesterasa/metabolismo , Sitios de Unión , Biocatálisis , Dominio Catalítico , Reactivadores de la Colinesterasa/metabolismo , Cristalografía por Rayos X , Humanos , Simulación de Dinámica Molecular , Compuestos Organotiofosforados/metabolismo , Oximas/metabolismo , Compuestos de Piridinio/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , EstereoisomerismoRESUMEN
Mycobacterium tuberculosis (Mtb) encodes two CRP/FNR family transcription factors (TF) that contribute to virulence, Cmr (Rv1675c) and CRPMt (Rv3676). Prior studies identified distinct chromosomal binding profiles for each TF despite their recognizing overlapping DNA motifs. The present study shows that Cmr binding specificity is determined by discriminator nucleotides at motif positions 4 and 13. X-ray crystallography and targeted mutational analyses identified an arginine-rich loop that expands Cmr's DNA interactions beyond the classical helix-turn-helix contacts common to all CRP/FNR family members and facilitates binding to imperfect DNA sequences. Cmr binding to DNA results in a pronounced asymmetric bending of the DNA and its high level of cooperativity is consistent with DNA-facilitated dimerization. A unique N-terminal extension inserts between the DNA binding and dimerization domains, partially occluding the site where the canonical cAMP binding pocket is found. However, an unstructured region of this N-terminus may help modulate Cmr activity in response to cellular signals. Cmr's multiple levels of DNA interaction likely enhance its ability to integrate diverse gene regulatory signals, while its novel structural features establish Cmr as an atypical CRP/FNR family member.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN/metabolismo , Secuencias Hélice-Giro-Hélice , Mycobacterium tuberculosis/metabolismo , Motivos de Nucleótidos , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión , Cristalografía por Rayos X , ADN/química , ADN/genética , Modelos Moleculares , Mycobacterium tuberculosis/genética , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
Malaria is a devastating disease in sub-Saharan Africa and is transmitted by the mosquito Anopheles gambiae. While indoor residual spraying of anticholinesterase insecticides has been useful in controlling the spread of malaria, widespread application of these compounds has led to the rise of an insecticide-resistant mosquito strain that harbors a G119S mutation in the nervous system target enzyme acetylcholinesterase. We demonstrate the atomic basis of insecticide resistance through structure determination of the G119S mutant acetylcholinesterase of An. gambiae in the ligand-free state and bound to a potent difluoromethyl ketone inhibitor. These structures reveal specific features within the active-site gorge distinct from human acetylcholinesterase, including an open channel at the base of the gorge, and provide a means for improving species selectivity in the rational design of improved insecticides for malaria vector control.
Asunto(s)
Acetilcolinesterasa/química , Anopheles/química , Inhibidores de la Colinesterasa/química , Fluoruros/química , Proteínas de Insectos/química , Insecticidas/química , Cetonas/química , Acetilcolinesterasa/genética , Acetilcolinesterasa/metabolismo , Sustitución de Aminoácidos , Animales , Anopheles/enzimología , Anopheles/genética , Baculoviridae/genética , Baculoviridae/metabolismo , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Proteínas de Insectos/antagonistas & inhibidores , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Resistencia a los Insecticidas/genética , Modelos Moleculares , Mosquitos Vectores/química , Mosquitos Vectores/enzimología , Mosquitos Vectores/genética , Mutación , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Especificidad de la Especie , SpodopteraRESUMEN
The monogenic and monoallelic expression of only one out of >1000 mouse olfactory receptor (ORs) genes requires the formation of large heterochromatic chromatin domains that sequester the OR gene clusters. Within these domains, intergenic transcriptional enhancers evade heterochromatic silencing and converge into interchromosomal hubs that assemble over the transcriptionally active OR. The significance of this nuclear organization in OR choice remains elusive. Here, we show that transcription factors Lhx2 and Ebf specify OR enhancers by binding in a functionally cooperative fashion to stereotypically spaced motifs that defy heterochromatin. Specific displacement of Lhx2 and Ebf from OR enhancers resulted in pervasive, long-range, and trans downregulation of OR transcription, whereas pre-assembly of a multi-enhancer hub increased the frequency of OR choice in cis. Our data provide genetic support for the requirement and sufficiency of interchromosomal interactions in singular OR choice and generate general regulatory principles for stochastic, mutually exclusive gene expression programs.
Asunto(s)
Regulación de la Expresión Génica , Proteínas con Homeodominio LIM/metabolismo , Neuronas/fisiología , Receptores Odorantes/biosíntesis , Receptores Odorantes/genética , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Animales , Ratones , Unión Proteica , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
Natural product inhibitors of AChE are of interest both because they offer promise as inexpensive drugs for symptomatic relief in Alzheimer's disease and because they may provide insights into the structural features of the AChE catalytic site. Hopeahainol A is an uncharged polyphenol AChE inhibitor from the stem bark of Hopea hainanensis with a constrained, partially dearomatized bicyclic core. Molecular modeling indicates that hopeahainol A binds at the entrance of the long but narrow AChE active site gorge because it is too bulky to be accommodated within the gorge without severe distortion of the gorge as depicted in AChE crystal structures. We conducted inhibitor competition experiments in which AChE inhibition was measured with hopeahainol A together with either edrophonium (which binds at the base of the gorge) or thioflavin T (which binds to the peripheral or P-site near the gorge mouth). The results agreed with the molecular modeling and indicated that hopeahainol A at lower concentrations (<200 µM) bound only to the P-site, as hopeahainol A and thioflavin T were unable to form a ternary complex with AChE while hopeahainol A and edrophonium did form a ternary complex with essentially no competition between them. Inhibition increased to a striking extent at higher concentrations of hopeahainol A, with plots analogous to classic Dixon plots showing a dependence on hopeahainol A concentrations to the third- or fourth order. The inhibition at higher hopeahainol A concentrations was completely reversed on dilution and blocked by bound edrophonium. We hypothesize that bound hopeahainol A induces conformational changes in the AChE active site that allow binding of additional hopeahainol A molecules, a phenomenon that would be unprecedented for a reversible inhibitor that apparently forms no covalent bonds with AChE.
Asunto(s)
Acetilcolinesterasa/metabolismo , Inhibidores de la Colinesterasa/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/metabolismo , Acetilcolinesterasa/química , Benzotiazoles , Sitios de Unión , Dominio Catalítico , Inhibidores de la Colinesterasa/química , Dipterocarpaceae/química , Dipterocarpaceae/metabolismo , Edrofonio/química , Edrofonio/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/química , Cinética , Simulación del Acoplamiento Molecular , Corteza de la Planta/química , Corteza de la Planta/metabolismo , Especificidad por Sustrato , Termodinámica , Tiazoles/química , Tiazoles/metabolismoRESUMEN
Irreversible inhibition of the essential nervous system enzyme acetylcholinesterase by organophosphate nerve agents and pesticides may quickly lead to death. Oxime reactivators currently used as antidotes are generally less effective against pesticide exposure than nerve agent exposure, and pesticide exposure constitutes the majority of cases of organophosphate poisoning in the world. The current lack of published structural data specific to human acetylcholinesterase organophosphate-inhibited and oxime-bound states hinders development of effective medical treatments. We have solved structures of human acetylcholinesterase in different states in complex with the organophosphate insecticide, paraoxon, and oximes. Reaction with paraoxon results in a highly perturbed acyl loop that causes a narrowing of the gorge in the peripheral site that may impede entry of reactivators. This appears characteristic of acetylcholinesterase inhibition by organophosphate insecticides but not nerve agents. Additional changes seen at the dimer interface are novel and provide further examples of the disruptive effect of paraoxon. Ternary structures of paraoxon-inhibited human acetylcholinesterase in complex with the oximes HI6 and 2-PAM reveals relatively poor positioning for reactivation. This study provides a structural foundation for improved reactivator design for the treatment of organophosphate intoxication. Proteins 2016; 84:1246-1256. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Acetilcolinesterasa/química , Antídotos/química , Inhibidores de la Colinesterasa/química , Insecticidas/química , Paraoxon/química , Compuestos de Pralidoxima/química , Acetilcolinesterasa/genética , Acetilcolinesterasa/metabolismo , Secuencias de Aminoácidos , Sitios de Unión , Línea Celular , Cristalografía por Rayos X , Células Epiteliales/citología , Células Epiteliales/enzimología , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Expresión Génica , Humanos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The Venezuelan equine encephalitis virus (VEEV) nonstructural protein 2 (nsP2) cysteine protease (EC 3.4.22.-) is essential for viral replication and is involved in the cytopathic effects (CPE) of the virus. The VEEV nsP2 protease is a member of MEROPS Clan CN and characteristically contains a papain-like protease linked to an S-adenosyl-l-methionine-dependent RNA methyltransferase (SAM MTase) domain. The protease contains an alternative active site motif, (475)NVCWAK(480), which differs from papain's (CGS(25)CWAFS), and the enzyme lacks a transition state-stabilizing residue homologous to Gln-19 in papain. To understand the roles of conserved residues in catalysis, we determined the structure of the free enzyme and the first structure of an inhibitor-bound alphaviral protease. The peptide-like E64d inhibitor was found to bind beneath a ß-hairpin at the interface of the SAM MTase and protease domains. His-546 adopted a conformation that differed from that found in the free enzyme; one or both of the conformers may assist in leaving group departure of either the amine or Cys thiolate during the catalytic cycle. Interestingly, E64c (200 µM), the carboxylic acid form of the E64d ester, did not inhibit the nsP2 protease. To identify key residues involved in substrate binding, a number of mutants were analyzed. Mutation of the motif residue, N475A, led to a 24-fold reduction in kcat/Km, and the conformation of this residue did not change after inhibition. N475 forms a hydrogen bond with R662 in the SAM MTase domain, and the R662A and R662K mutations both led to 16-fold decreases in kcat/Km. N475 forms the base of the P1 binding site and likely orients the substrate for nucleophilic attack or plays a role in product release. An Asn homologous to N475 is similarly found in coronaviral papain-like proteases (PLpro) of the Severe Acute Respiratory Syndrome (SARS) virus and Middle East Respiratory Syndrome (MERS) virus. Mutation of another motif residue, K480A, led to a 9-fold decrease in kcat and kcat/Km. K480 likely enhances the nucleophilicity of the Cys. Consistent with our substrate-bound models, the SAM MTase domain K706A mutation increased Km 4.5-fold to 500 µM. Within the ß-hairpin, the N545A mutation slightly but not significantly increased kcat and Km. The structures and identified active site residues may facilitate the discovery of protease inhibitors with antiviral activity.
Asunto(s)
Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Virus de la Encefalitis Equina Venezolana/enzimología , Mutación/genética , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Cisteína Endopeptidasas/metabolismo , Hidrólisis , Cinética , Modelos Moleculares , Papaína/metabolismo , Conformación Proteica , S-Adenosilmetionina/metabolismo , Homología de Secuencia de Aminoácido , Proteínas no Estructurales Virales/metabolismoRESUMEN
Coxiella burnetii is a highly infectious bacterium and potential agent of bioterrorism. However, it has not been studied as extensively as other biological agents, and very few of its proteins have been structurally characterized. To address this situation, we undertook a study of critical metabolic enzymes in C. burnetii that have great potential as drug targets. We used high-throughput techniques to produce novel crystal structures of 48 of these proteins. We selected one protein, C. burnetii dihydrofolate reductase (CbDHFR), for additional work to demonstrate the value of these structures for structure-based drug design. This enzyme's structure reveals a feature in the substrate binding groove that is different between CbDHFR and human dihydrofolate reductase (hDHFR). We then identified a compound by in silico screening that exploits this binding groove difference, and demonstrated that this compound inhibits CbDHFR with at least 25-fold greater potency than hDHFR. Since this binding groove feature is shared by many other prokaryotes, the compound identified could form the basis of a novel antibacterial agent effective against a broad spectrum of pathogenic bacteria.
Asunto(s)
Proteínas Bacterianas/química , Coxiella burnetii/efectos de los fármacos , Coxiella burnetii/genética , Antagonistas del Ácido Fólico/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Simulación por Computador , Cristalografía por Rayos X , Diseño de Fármacos , Antagonistas del Ácido Fólico/química , Humanos , Conformación Proteica , Tetrahidrofolato Deshidrogenasa/químicaRESUMEN
The extensively studied cAMP-dependent protein kinase A (PKA) is involved in the regulation of critical cell processes, including metabolism, gene expression, and cell proliferation; consequentially, mis-regulation of PKA signaling is implicated in tumorigenesis. Recent genomic studies have identified recurrent mutations in the catalytic subunit of PKA in tumors associated with Cushing's syndrome, a kidney disorder leading to excessive cortisol production, and also in tumors associated with fibrolamellar hepatocellular carcinoma (FL-HCC), a rare liver cancer. Expression of a L205R point mutant and a DnaJ-PKA fusion protein were found to be linked to Cushing's syndrome and FL-HCC, respectively. Here we reveal contrasting mechanisms for increased PKA signaling at the molecular level through structural determination and biochemical characterization of the aberrant enzymes. In the Cushing's syndrome disorder, we find that the L205R mutation abolishes regulatory-subunit binding, leading to constitutive, cAMP-independent signaling. In FL-HCC, the DnaJ-PKA chimera remains under regulatory subunit control; however, its overexpression from the DnaJ promoter leads to enhanced cAMP-dependent signaling. Our findings provide a structural understanding of the two distinct disease mechanisms and they offer a basis for designing effective drugs for their treatment.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Neoplasias/enzimología , Dominio Catalítico , Cromatografía en Gel , Cristalización , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Dimerización , Humanos , Modelos Moleculares , Mutación , Conformación Proteica , Resonancia por Plasmón de SuperficieRESUMEN
Ricin is a select agent toxin and a member of the RNA N-glycosidase family of medically important plant and bacterial ribosome-inactivating proteins. In this study, we determined X-ray crystal structures of the enzymatic subunit of ricin (RTA) in complex with the antigen binding domains (VHH) of five unique single-chain monoclonal antibodies that differ in their respective toxin-neutralizing activities. None of the VHHs made direct contact with residues involved in RTA's RNA N-glycosidase activity or induced notable allosteric changes in the toxin's subunit. Rather, the five VHHs had overlapping structural epitopes on the surface of the toxin and differed in the degree to which they made contact with prominent structural elements in two folding domains of the RTA. In general, RTA interactions were influenced most by the VHH CDR3 (CDR, complementarity-determining region) elements, with the most potent neutralizing antibody having the shortest and most conformationally constrained CDR3. These structures provide unique insights into the mechanisms underlying toxin neutralization and provide critically important information required for the rational design of ricin toxin subunit vaccines.
Asunto(s)
Anticuerpos Neutralizantes/química , Ricina/química , Anticuerpos de Cadena Única/química , Secuencia de Aminoácidos , Animales , Camelus , Ricinus communis/enzimología , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Ricina/antagonistas & inhibidoresRESUMEN
Acetylcholinesterase (AChE) is a critical enzyme that regulates neurotransmission by degrading the neurotransmitter acetylcholine in synapses of the nervous system. It is an important target for both therapeutic drugs that treat Alzheimer's disease and organophosphate (OP) chemical warfare agents that cripple the nervous system and cause death through paralysis. We are exploring a strategy to design compounds that bind tightly at or near a peripheral or P-site near the mouth of the AChE active site gorge and exclude OPs from the active site while interfering minimally with the passage of acetylcholine. However, to target the AChE P-site, much more information must be gathered about the structure-activity relationships of ligands that bind specifically to the P-site. Here, we review our recent reports on two uncharged, natural product inhibitors of AChE, dihydrotanshinone I and territrem B, that have relatively high affinities for the enzyme. We describe an inhibitor competition assay and comment on the structures of these inhibitors in complex with recombinant human acetylcholinesterase as determined by X-ray crystallography. Our results reveal that dihydrotanshinone I binding is specific to only the P-site, while territrem B binding spans the P-site and extends into the acylation or A-site at the base of the gorge.
Asunto(s)
Acetilcolinesterasa/química , Productos Biológicos/farmacología , Inhibidores de la Colinesterasa/farmacología , Fenantrenos/farmacología , Piranos/farmacología , Acetilcolinesterasa/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Productos Biológicos/química , Inhibidores de la Colinesterasa/química , Furanos , Humanos , Datos de Secuencia Molecular , Fenantrenos/química , Piranos/química , QuinonasRESUMEN
Cholinergic synaptic transmission often requires extremely rapid hydrolysis of acetylcholine by acetylcholinesterase (AChE). AChE is inactivated by organophosphates (OPs) in chemical warfare nerve agents. The resulting accumulation of acetylcholine disrupts cholinergic synaptic transmission and can lead to death. A potential long-term strategy for preventing AChE inactivation by OPs is based on evidence that OPs must pass through a peripheral site or P-site near the mouth of the AChE active site gorge before reacting with a catalytic serine in an acylation site or A-site at the base of the gorge. An ultimate goal of this strategy is to design compounds that bind tightly at or near the P-site and exclude OPs from the active site while interfering minimally with the passage of acetylcholine. However, to target the AChE P-site with ligands and potential drugs that selectively restrict access, much more information must be gathered about the structure-activity relationships of ligands that bind specifically to the P-site. We apply here an inhibitor competition assay that can correctly determine whether an AChE inhibitor binds to the P-site, the A-site, or both sites. We have used this assay to examine three uncharged, natural product inhibitors of AChE, including aflatoxin B1, dihydrotanshinone I, and territrem B. The first two of these inhibitors are predicted by the competition assay to bind selectively to the P-site, while territrem B is predicted to span both the P- and A-sites. These predictions have recently been confirmed by X-ray crystallography. Dihydrotanshinone I, with an observed binding constant (KI) of 750 nM, provides a good lead compound for the development of high-affinity, uncharged inhibitors with specificity for the P-site.
Asunto(s)
Acetilcolinesterasa/química , Acetilcolinesterasa/metabolismo , Inhibidores de la Colinesterasa/farmacología , Fenantrenos/farmacología , Acetilcolina/metabolismo , Acetilcolinesterasa/genética , Aflatoxina B1/farmacología , Sitios de Unión , Unión Competitiva , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Furanos , Humanos , Hidrólisis , Cinética , Modelos Químicos , Piranos/farmacología , Quinonas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
In the future, we may be faced with the need to provide treatment for an emergent biological threat against which existing vaccines and drugs have limited efficacy or availability. To prepare for this eventuality, our objective was to use a metabolic network-based approach to rapidly identify potential drug targets and prospectively screen and validate novel small-molecule antimicrobials. Our target organism was the fully virulent Francisella tularensis subspecies tularensis Schu S4 strain, a highly infectious intracellular pathogen that is the causative agent of tularemia and is classified as a category A biological agent by the Centers for Disease Control and Prevention. We proceeded with a staggered computational and experimental workflow that used a strain-specific metabolic network model, homology modeling and X-ray crystallography of protein targets, and ligand- and structure-based drug design. Selected compounds were subsequently filtered based on physiological-based pharmacokinetic modeling, and we selected a final set of 40 compounds for experimental validation of antimicrobial activity. We began screening these compounds in whole bacterial cell-based assays in biosafety level 3 facilities in the 20th week of the study and completed the screens within 12 weeks. Six compounds showed significant growth inhibition of F. tularensis, and we determined their respective minimum inhibitory concentrations and mammalian cell cytotoxicities. The most promising compound had a low molecular weight, was non-toxic, and abolished bacterial growth at 13 µM, with putative activity against pantetheine-phosphate adenylyltransferase, an enzyme involved in the biosynthesis of coenzyme A, encoded by gene coaD. The novel antimicrobial compounds identified in this study serve as starting points for lead optimization, animal testing, and drug development against tularemia. Our integrated in silico/in vitro approach had an overall 15% success rate in terms of active versus tested compounds over an elapsed time period of 32 weeks, from pathogen strain identification to selection and validation of novel antimicrobial compounds.
Asunto(s)
Antibacterianos/farmacología , Descubrimiento de Drogas , Francisella tularensis/efectos de los fármacos , Francisella tularensis/metabolismo , Redes y Vías Metabólicas/efectos de los fármacos , Antibacterianos/química , Antibacterianos/farmacocinética , Proteínas Bacterianas/química , Simulación por Computador , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Humanos , Cinética , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacosRESUMEN
Acetylcholinesterase is a critical enzyme that regulates neurotransmission by degrading the neurotransmitter acetylcholine in synapses of the nervous system. It is an important target for both therapeutic drugs that treat Alzheimer's disease and chemical warfare agents that cripple the nervous system and cause death through paralysis. The enzyme has both catalytic and peripheral sites to which inhibitors may bind. Structures of recombinant human acetylcholinesterase in complex with the natural product inhibitors dihydrotanshinone I and territrem B reveal dihydrotanshinone I binding that is specific to only the peripheral site and territrem B binding that spans both sites and distorts the protein backbone in the peripheral site. These inhibitors may function as important molecular templates for therapeutics used for treatment of disease and protection against nerve agents.