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1.
Proc Natl Acad Sci U S A ; 116(9): 3853-3862, 2019 02 26.
Artículo en Inglés | MEDLINE | ID: mdl-30755521

RESUMEN

The human dopamine (DA) transporter (hDAT) mediates clearance of DA. Genetic variants in hDAT have been associated with DA dysfunction, a complication associated with several brain disorders, including autism spectrum disorder (ASD). Here, we investigated the structural and behavioral bases of an ASD-associated in-frame deletion in hDAT at N336 (∆N336). We uncovered that the deletion promoted a previously unobserved conformation of the intracellular gate of the transporter, likely representing the rate-limiting step of the transport process. It is defined by a "half-open and inward-facing" state (HOIF) of the intracellular gate that is stabilized by a network of interactions conserved phylogenetically, as we demonstrated in hDAT by Rosetta molecular modeling and fine-grained simulations, as well as in its bacterial homolog leucine transporter by electron paramagnetic resonance analysis and X-ray crystallography. The stabilization of the HOIF state is associated both with DA dysfunctions demonstrated in isolated brains of Drosophila melanogaster expressing hDAT ∆N336 and with abnormal behaviors observed at high-time resolution. These flies display increased fear, impaired social interactions, and locomotion traits we associate with DA dysfunction and the HOIF state. Together, our results describe how a genetic variation causes DA dysfunction and abnormal behaviors by stabilizing a HOIF state of the transporter.


Asunto(s)
Trastorno del Espectro Autista/genética , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Dopamina/genética , Locomoción/genética , Animales , Animales Modificados Genéticamente , Trastorno del Espectro Autista/fisiopatología , Cristalografía por Rayos X , Dopamina/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/química , Drosophila melanogaster/genética , Drosophila melanogaster/fisiología , Espectroscopía de Resonancia por Spin del Electrón , Miedo/fisiología , Humanos , Relaciones Interpersonales , Locomoción/fisiología , Modelos Moleculares , Mutación , Eliminación de Secuencia/genética
2.
Acta Crystallogr D Biol Crystallogr ; 71(Pt 5): 1216-25, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25945586

RESUMEN

Homoserine dehydrogenase (HSD) is an oxidoreductase in the aspartic acid pathway. This enzyme coordinates a critical branch point of the metabolic pathway that leads to the synthesis of bacterial cell-wall components such as L-lysine and m-DAP in addition to other amino acids such as L-threonine, L-methionine and L-isoleucine. Here, a structural rationale for the hydride-transfer step in the reaction mechanism of HSD is reported. The structure of Staphylococcus aureus HSD was determined at different pH conditions to understand the basis for the enhanced enzymatic activity at basic pH. An analysis of the crystal structure revealed that Lys105, which is located at the interface of the catalytic and cofactor-binding sites, could mediate the hydride-transfer step of the reaction mechanism. The role of Lys105 was subsequently confirmed by mutational analysis. Put together, these studies reveal the role of conserved water molecules and a lysine residue in hydride transfer between the substrate and the cofactor.


Asunto(s)
Homoserina Deshidrogenasa/química , Homoserina Deshidrogenasa/metabolismo , Lisina/química , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Staphylococcus aureus/enzimología , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Homoserina Deshidrogenasa/genética , Cinética , Lisina/genética , Lisina/metabolismo , Modelos Moleculares , Proteínas Mutantes/genética , Mutación/genética , Unión Proteica , Conformación Proteica
3.
bioRxiv ; 2024 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-38187772

RESUMEN

In C. elegans, inter-cellular transport of the small non-coding RNA causing systemic RNA interference (RNAi) is mediated by the transmembrane protein SID1, encoded by the sid1 gene in the systemic RNA interference-defective (sid) loci. SID1 shares structural and sequence similarity with cholesterol uptake protein 1 (CHUP1) and is classified as a member of the cholesterol uptake family (ChUP). Although systemic RNAi is not an evolutionarily conserved process, the sid gene products are found across the animal kingdom, suggesting the existence of other novel gene regulatory mechanisms mediated by small non-coding RNAs. Human homologs of sid gene products - hSIDT1 and hSIDT2 - mediate contact-dependent lipophilic small non-coding dsRNA transport. Here, we report the structure of recombinant human SIDT1. We find that the extra-cytosolic domain (ECD) of hSIDT1 adopts a double jelly roll fold, and the transmembrane domain (TMD) exists as two modules - a flexible lipid binding domain (LBD) and a rigid TMD core. Our structural analyses provide insights into the inherent conformational dynamics within the lipid binding domain in cholesterol uptake (ChUP) family members.

4.
Life Sci Alliance ; 7(9)2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-38925866

RESUMEN

In Caenorhabditis elegans, inter-cellular transport of the small non-coding RNA causing systemic RNAi is mediated by the transmembrane protein SID1, encoded by the sid1 gene in the systemic RNAi defective (sid) loci. SID1 shares structural and sequence similarity with cholesterol uptake protein 1 (CHUP1) and is classified as a member of the ChUP family. Although systemic RNAi is not an evolutionarily conserved process, the sid gene products are found across the animal kingdom, suggesting the existence of other novel gene regulatory mechanisms mediated by small non-coding RNAs. Human homologs of sid gene products-hSIDT1 and hSIDT2-mediate contact-dependent lipophilic small non-coding dsRNA transport. Here, we report the structure of recombinant human SIDT1. We find that the extra-cytosolic domain of hSIDT1 adopts a double jelly roll fold, and the transmembrane domain exists as two modules-a flexible lipid binding domain and a rigid transmembrane domain core. Our structural analyses provide insights into the inherent conformational dynamics within the lipid binding domain in ChUP family members.


Asunto(s)
Proteínas de la Membrana , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/química , Unión Proteica , Dominios Proteicos/genética , Modelos Moleculares , Conformación Proteica , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Animales , Secuencia de Aminoácidos , Sitios de Unión , Lípidos/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Interferencia de ARN
5.
Artículo en Inglés | MEDLINE | ID: mdl-24192352

RESUMEN

Staphylococcus aureus is a Gram-positive nosocomial pathogen. The prevalence of multidrug-resistant S. aureus strains in both hospital and community settings makes it imperative to characterize new drug targets to combat S. aureus infections. In this context, enzymes involved in cell-wall maintenance and essential amino-acid biosynthesis are significant drug targets. Homoserine dehydrogenase (HSD) is an oxidoreductase that is involved in the reversible conversion of L-aspartate semialdehyde to L-homoserine in a dinucleotide cofactor-dependent reduction reaction. HSD is thus a crucial intermediate enzyme linked to the biosynthesis of several essential amino acids such as lysine, methionine, isoleucine and threonine.


Asunto(s)
Proteínas Bacterianas/química , Homoserina Deshidrogenasa/química , Staphylococcus aureus/enzimología , Proteínas Bacterianas/aislamiento & purificación , Cristalización , Homoserina Deshidrogenasa/aislamiento & purificación , Concentración de Iones de Hidrógeno , Temperatura , Difracción de Rayos X
6.
bioRxiv ; 2023 Oct 26.
Artículo en Inglés | MEDLINE | ID: mdl-37961489

RESUMEN

Degradation of heparan sulfate (HS), a glycosaminoglycan (GAG) comprised of repeating units of N-acetylglucosamine and glucuronic acid, begins in the cytosol and is completed in the lysosomes. Acetylation of the terminal non-reducing amino group of α-D-glucosamine of HS is essential for its complete breakdown into monosaccharides and free sulfate. Heparan-α-glucosaminide N-acetyltransferase (HGSNAT), a resident of the lysosomal membrane, catalyzes this essential acetylation reaction by accepting and transferring the acetyl group from cytosolic acetyl-CoA to terminal α-D-glucosamine of HS in the lysosomal lumen. Mutation-induced dysfunction in HGSNAT causes abnormal accumulation of HS within the lysosomes and leads to an autosomal recessive neurodegenerative lysosomal storage disorder called mucopolysaccharidosis IIIC (MPS IIIC). There are no approved drugs or treatment strategies to cure or manage the symptoms of, MPS IIIC. Here, we use cryo-electron microscopy (cryo-EM) to determine a high-resolution structure of the HGSNAT-acetyl-CoA complex in an open-to-lumen conformation, the first step in HGSNAT catalyzed acetyltransferase reaction. In addition, we map the known MPS IIIC mutations onto the structure and elucidate the molecular basis for mutation-induced HGSNAT dysfunction.

7.
J Bacteriol ; 192(1): 134-44, 2010 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-19854906

RESUMEN

Penicillin binding proteins (PBPs) are membrane-associated proteins that catalyze the final step of murein biosynthesis. These proteins function as either transpeptidases or carboxypeptidases and in a few cases demonstrate transglycosylase activity. Both transpeptidase and carboxypeptidase activities of PBPs occur at the D-Ala-D-Ala terminus of a murein precursor containing a disaccharide pentapeptide comprising N-acetylglucosamine and N-acetyl-muramic acid-L-Ala-D-Glu-L-Lys-D-Ala-D-Ala. Beta-lactam antibiotics inhibit these enzymes by competing with the pentapeptide precursor for binding to the active site of the enzyme. Here we describe the crystal structure, biochemical characteristics, and expression profile of PBP4, a low-molecular-mass PBP from Staphylococcus aureus strain COL. The crystal structures of PBP4-antibiotic complexes reported here were determined by molecular replacement, using the atomic coordinates deposited by the New York Structural Genomics Consortium. While the pbp4 gene is not essential for the viability of S. aureus, the knockout phenotype of this gene is characterized by a marked reduction in cross-linked muropeptide and increased vancomycin resistance. Unlike other PBPs, we note that expression of PBP4 was not substantially altered under different experimental conditions, nor did it change across representative hospital- or community-associated strains of S. aureus that were examined. In vitro data on purified recombinant S. aureus PBP4 suggest that it is a beta-lactamase and is not trapped as an acyl intermediate with beta-lactam antibiotics. Put together, the expression analysis and biochemical features of PBP4 provide a framework for understanding the function of this protein in S. aureus and its role in antimicrobial resistance.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana/fisiología , Proteínas de Unión a las Penicilinas/química , Proteínas de Unión a las Penicilinas/metabolismo , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/metabolismo , Ampicilina/farmacología , Proteínas Bacterianas/genética , Cefotaxima/farmacología , Cristalografía por Rayos X , Datos de Secuencia Molecular , Oxacilina/farmacología , Proteínas de Unión a las Penicilinas/genética , Reacción en Cadena de la Polimerasa , Resistencia a la Vancomicina , beta-Lactamas/farmacología
8.
Elife ; 92020 07 03.
Artículo en Inglés | MEDLINE | ID: mdl-32618269

RESUMEN

Antidepressants target the serotonin transporter (SERT) by inhibiting serotonin reuptake. Structural and biochemical studies aiming to understand binding of small-molecules to conformationally dynamic transporters like SERT often require thermostabilizing mutations and antibodies to stabilize a specific conformation, leading to questions about relationships of these structures to the bonafide conformation and inhibitor binding poses of wild-type transporter. To address these concerns, we determined the structures of ∆N72/∆C13 and ts2-inactive SERT bound to paroxetine analogues using single-particle cryo-EM and x-ray crystallography, respectively. We synthesized enantiopure analogues of paroxetine containing either bromine or iodine instead of fluorine. We exploited the anomalous scattering of bromine and iodine to define the pose of these inhibitors and investigated inhibitor binding to Asn177 mutants of ts2-active SERT. These studies provide mutually consistent insights into how paroxetine and its analogues bind to the central substrate-binding site of SERT, stabilize the outward-open conformation, and inhibit serotonin transport.


Asunto(s)
Paroxetina/química , Inhibidores Selectivos de la Recaptación de Serotonina/química , Proteínas de Transporte de Serotonina en la Membrana Plasmática/química , Microscopía por Crioelectrón , Cristalografía por Rayos X , Humanos , Estructura Molecular , Paroxetina/metabolismo , Estructura Terciaria de Proteína , Serotonina/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Inhibidores Selectivos de la Recaptación de Serotonina/metabolismo
9.
Curr Opin Struct Biol ; 54: 161-170, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30921707

RESUMEN

Neurotransmitter sodium symporters (NSS) belong to the SLC6 family of solute carriers and play an essential role in neurotransmitter homeostasis throughout the body. In the past decade, structural studies employing bacterial orthologs of NSSs have provided insight into the mechanism of neurotransmitter transport. While the overall architecture of SLC6 transporters is conserved among species, in comparison to the bacterial homologs, the eukaryotic SLC6 family members harbor differences in amino acid sequence and molecular structure, which underpins their functional and pharmacological diversity, as well as their ligand specificity. Here, we review the structures and mechanisms of eukaryotic NSSs, focusing on the molecular basis for ligand recognition and on transport mechanism.


Asunto(s)
Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/metabolismo , Animales , Sitios de Unión , Secuencia Conservada , Humanos , Terapia Molecular Dirigida , Farmacología , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/antagonistas & inhibidores , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/química
10.
PLoS One ; 13(7): e0200085, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29965988

RESUMEN

The human dopamine transporter (hDAT) plays a major role in dopamine homeostasis and regulation of neurotransmission by clearing dopamine from the extracellular space using secondary active transport. Dopamine is an essential monoamine chemical messenger that regulates reward seeking behavior, motor control, hormonal release, and emotional response in humans. Psychostimulants such as cocaine primarily target the central binding site of hDAT and lock the transporter in an outward-facing conformation, thereby inhibiting dopamine reuptake. The inhibition of dopamine reuptake leads to accumulation of dopamine in the synapse causing heightened signaling. In addition, hDAT is implicated in various neurological disorders and disease-associated neurodegeneration. Despite its significance, the structural studies of hDAT have proven difficult. Instability of hDAT in detergent micelles has been a limiting factor in its successful biochemical, biophysical, and structural characterization. To overcome this hurdle, we identified ligands that stabilize hDAT in detergent micelles. We then screened ~200 single residue mutants of hDAT using a high-throughput scintillation proximity assay and identified a thermostable variant (I248Y). Here we report a robust strategy to overexpress and successfully purify a thermostable variant of hDAT in an inhibitor and allosteric ligand bound conformation.


Asunto(s)
Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/química , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/aislamiento & purificación , Regulación Alostérica , Membrana Celular/metabolismo , Dopamina/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/antagonistas & inhibidores , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Ligandos , Micelas , Mutagénesis Sitio-Dirigida , Mutación , Conformación Proteica , Estabilidad Proteica , Temperatura
11.
Sci Rep ; 6: 30827, 2016 08 09.
Artículo en Inglés | MEDLINE | ID: mdl-27501775

RESUMEN

The Mycobacterium tuberculosis dihydrodipicolinate synthase (Mtb-dapA) is an essential gene. Mtb-DapA catalyzes the aldol condensation between pyruvate and L-aspartate-beta-semialdehyde (ASA) to yield dihydrodipicolinate. In this work we tested the inhibitory effects of structural analogues of pyruvate on recombinant Mtb-DapA (Mtb-rDapA) using a coupled assay with recombinant dihydrodipicolinate reductase (Mtb-rDapB). Alpha-ketopimelic acid (α-KPA) showed maximum inhibition of 88% and IC50 of 21 µM in the presence of pyruvate (500 µM) and ASA (400 µM). Competition experiments with pyruvate and ASA revealed competition of α-KPA with pyruvate. Liquid chromatography-mass spectrometry (LC-MS) data with multiple reaction monitoring (MRM) showed that the relative abundance peak of final product, 2,3,4,5-tetrahydrodipicolinate, was decreased by 50%. Thermal shift assays showed 1 °C Tm shift of Mtb-rDapA upon binding α-KPA. The 2.4 Å crystal structure of Mtb-rDapA-α-KPA complex showed the interaction of critical residues at the active site with α-KPA. Molecular dynamics simulations over 500 ns of pyruvate docked to Mtb-DapA and of α-KPA-bound Mtb-rDapA revealed formation of hydrogen bonds with pyruvate throughout in contrast to α-KPA. Molecular descriptors analysis showed that ligands with polar surface area of 91.7 Å(2) are likely inhibitors. In summary, α-hydroxypimelic acid and other analogues could be explored further as inhibitors of Mtb-DapA.


Asunto(s)
Proteínas Bacterianas/metabolismo , Hidrocarburos Aromáticos con Puentes/farmacología , Hidroliasas/metabolismo , Cetonas/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Ácido Aspártico/análogos & derivados , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Sitios de Unión , Hidrocarburos Aromáticos con Puentes/química , Hidrocarburos Aromáticos con Puentes/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Hidroliasas/antagonistas & inhibidores , Hidroliasas/genética , Enlace de Hidrógeno , Concentración 50 Inhibidora , Cetonas/química , Cetonas/metabolismo , Cinética , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Ácido Pirúvico/química , Ácido Pirúvico/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación
12.
Bioelectrochemistry ; 98: 46-52, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24691485

RESUMEN

The use of titania nanotubes (TiO2-NT) as the working electrode provides a substantial improvement in the electrochemical detection of proteins. A biosensor designed using this strategy provided a robust method to detect protein samples at very low concentrations (Cprotein ca 1ng/µl). Reproducible measurements on protein samples at this concentration (Ip,a of 80+1.2µA) could be achieved using a sample volume of ca 30µl. We demonstrate the feasibility of this strategy for the accurate detection of penicillin binding protein, PBP2a, a marker for methicillin resistant Staphylococcus aureus (MRSA). The selectivity and efficiency of this sensor were also validated using other diverse protein preparations such as a recombinant protein tyrosine phosphatase (PTP10D) and bovine serum albumin (BSA). This electrochemical method also presents a substantial improvement in the time taken (few minutes) when compared to conventional enzyme-linked immunosorbent assay (ELISA) protocols. It is envisaged that this sensor could substantially aid in the rapid diagnosis of bacterial infections in resource strapped environments.


Asunto(s)
Técnicas Biosensibles/instrumentación , Carbono/química , Nanotubos/química , Proteínas/análisis , Titanio/química , Biomarcadores/análisis , Técnicas Biosensibles/métodos , Técnicas Electroquímicas , Electrodos , Estudios de Factibilidad , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Proteínas de Unión a las Penicilinas/análisis , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Factores de Tiempo
13.
FEBS Lett ; 585(16): 2561-7, 2011 Aug 19.
Artículo en Inglés | MEDLINE | ID: mdl-21803042

RESUMEN

Lysine biosynthesis proceeds by the nucleotide-dependent reduction of dihydrodipicolinate (DHDP) to tetrahydrodipicolinate (THDP) by dihydrodipicolinate reductase (DHDPR). The S. aureus DHDPR structure reveals different conformational states of this enzyme even in the absence of a substrate or nucleotide-cofactor. Despite lacking a conserved basic residue essential for NADPH interaction, S. aureus DHDPR differs from other homologues as NADPH is a more preferred co-factor than NADH. The structure provides a rationale-Lys35 compensates for the co-factor site mutation. These observations are significant for bi-ligand inhibitor design that relies on ligand-induced conformational changes as well as co-factor specificity for this important drug target.


Asunto(s)
Dihidrodipicolinato-Reductasa/química , Dihidrodipicolinato-Reductasa/metabolismo , NADP/metabolismo , Staphylococcus aureus/enzimología , Secuencia de Aminoácidos , Cristalografía por Rayos X , Dihidrodipicolinato-Reductasa/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Especificidad por Sustrato
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