RESUMEN
Gram negative pathogens are protected against toxic electrophilic compounds by glutathione-gated potassium efflux systems (Kef) that modulate cytoplasmic pH. We have elucidated the mechanism of gating through structural and functional analysis of Escherichia coli KefC. The revealed mechanism can explain how subtle chemical differences in glutathione derivatives can produce opposite effects on channel function. Kef channels are regulated by potassium transport and NAD-binding (KTN) domains that sense both reduced glutathione, which inhibits Kef activity, and glutathione adducts that form during electrophile detoxification and activate Kef. We find that reduced glutathione stabilizes an interdomain association between two KTN folds, whereas large adducts sterically disrupt this interaction. F441 is identified as the pivotal residue discriminating between reduced glutathione and its conjugates. We demonstrate a major structural change on the binding of an activating ligand to a KTN-domain protein. Analysis of the regulatory interactions suggests strategies to disrupt pathogen potassium and pH homeostasis.
Asunto(s)
Escherichia coli/metabolismo , Activación del Canal Iónico/fisiología , Potasio/metabolismo , Secuencia de Aminoácidos , Transporte Biológico/efectos de los fármacos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Glutatión/análogos & derivados , Glutatión/metabolismo , Glutatión/farmacología , Activación del Canal Iónico/efectos de los fármacos , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Canales de Potasio/química , Canales de Potasio/metabolismo , Unión Proteica/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína , Succinimidas/farmacologíaRESUMEN
Uridine phosphorylase (UPP) catalyzes the reversible conversion of uridine to uracil and ribose-1-phosphate and plays an important pharmacological role in activating fluoropyrimidine nucleoside chemotherapeutic agents such as 5-fluorouracil and capecitabine. Most vertebrate animals, including humans, possess two homologs of this enzyme (UPP1 & UPP2), of which UPP1 has been more thoroughly studied and is better characterized. Here, we report two crystallographic structures of human UPP2 (hUPP2) in distinctly active and inactive conformations. These structures reveal that a conditional intramolecular disulfide bridge can form within the protein that dislocates a critical phosphate-coordinating arginine residue (R100) away from the active site, disabling the enzyme. In vitro activity measurements on both recombinant hUPP2 and native mouse UPP2 confirm the redox sensitivity of this enzyme, in contrast to UPP1. Sequence analysis shows that this feature is conserved among UPP2 homologs and lacking in all UPP1 proteins due to the absence of a necessary cysteine residue. The state of the disulfide bridge has further structural consequences for one face of the enzyme that suggest UPP2 may have additional functions in sensing and initiating cellular responses to oxidative stress. The molecular details surrounding these dynamic aspects of hUPP2 structure and regulation provide new insights as to how novel inhibitors of this protein may be developed with improved specificity and affinity. As uridine is emerging as a promising protective compound in neuro-degenerative diseases, including Alzheimer's and Parkinson's, understanding the regulatory mechanisms underlying UPP control of uridine concentration is key to improving clinical outcomes in these illnesses.
Asunto(s)
Uridina Fosforilasa/química , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Cristalografía por Rayos X , Cistina/química , Pruebas de Enzimas , Humanos , Enlace de Hidrógeno , Ratones , Modelos Moleculares , Oxidación-Reducción , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Uracilo/análogos & derivados , Uracilo/química , Uridina Fosforilasa/antagonistas & inhibidoresRESUMEN
Obesity-associated insulin resistance plays a central role in the pathogenesis of type 2 diabetes. A promising approach to decrease insulin resistance in obesity is to inhibit the protein tyrosine phosphatases that negatively regulate insulin receptor signaling. The low-molecular-weight protein tyrosine phosphatase (LMPTP) acts as a critical promoter of insulin resistance in obesity by inhibiting phosphorylation of the liver insulin receptor activation motif. Here, we report development of a novel purine-based chemical series of LMPTP inhibitors. These compounds inhibit LMPTP with an uncompetitive mechanism and are highly selective for LMPTP over other protein tyrosine phosphatases. We also report the generation of a highly orally bioavailable purine-based analogue that reverses obesity-induced diabetes in mice.
Asunto(s)
Inhibidores Enzimáticos/química , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Purinas/química , Administración Oral , Animales , Sitios de Unión , Cristalografía por Rayos X , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/etiología , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Semivida , Humanos , Resistencia a la Insulina , Cinética , Simulación de Dinámica Molecular , Obesidad/complicaciones , Obesidad/patología , Fosforilación/efectos de los fármacos , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Purinas/metabolismo , Purinas/farmacología , Purinas/uso terapéutico , Transducción de Señal/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
BACKGROUND: Uridine phosphorylase (UPP) is a key enzyme of pyrimidine salvage pathways, catalyzing the reversible phosphorolysis of ribosides of uracil to nucleobases and ribose 1-phosphate. It is also a critical enzyme in the activation of pyrimidine-based chemotherapeutic compounds such a 5-fluorouracil (5-FU) and its prodrug capecitabine. Additionally, an elevated level of this enzyme in certain tumours is believed to contribute to the selectivity of such drugs. However, the clinical effectiveness of these fluoropyrimidine antimetabolites is hampered by their toxicity to normal tissue. In response to this limitation, specific inhibitors of UPP, such as 5-benzylacyclouridine (BAU), have been developed and investigated for their ability to modulate the cytotoxic side effects of 5-FU and its derivatives, so as to increase the therapeutic index of these agents. RESULTS: In this report we present the high resolution structures of human uridine phosphorylase 1 (hUPP1) in ligand-free and BAU-inhibited conformations. The structures confirm the unexpected solution observation that the human enzyme is dimeric in contrast to the hexameric assembly present in microbial UPPs. They also reveal in detail the mechanism by which BAU engages the active site of the protein and subsequently disables the enzyme by locking the protein in a closed conformation. The observed inter-domain motion of the dimeric human enzyme is much greater than that seen in previous UPP structures and may result from the simpler oligomeric organization. CONCLUSION: The structural details underlying hUPP1's active site and additional surfaces beyond these catalytic residues, which coordinate binding of BAU and other acyclouridine analogues, suggest avenues for future design of more potent inhibitors of this enzyme. Notably, the loop forming the back wall of the substrate binding pocket is conformationally different and substantially less flexible in hUPP1 than in previously studied microbial homologues. These distinctions can be utilized to discover novel inhibitory compounds specifically optimized for efficacy against the human enzyme as a step toward the development of more effective chemotherapeutic regimens that can selectively protect normal tissues with inherently lower UPP activity.
Asunto(s)
Inhibidores Enzimáticos/metabolismo , Uracilo/análogos & derivados , Uridina Fosforilasa/química , Uridina Fosforilasa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Dominio Catalítico , Cristalización , Dimerización , Diseño de Fármacos , Inhibidores Enzimáticos/química , Escherichia coli/genética , Humanos , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología Estructural de Proteína , Uracilo/química , Uracilo/metabolismo , Uridina Fosforilasa/antagonistas & inhibidores , Uridina Fosforilasa/genéticaRESUMEN
Recently, rapid progress in our structural knowledge of K(+)-selective channels has started to provide a basis for comprehending the biophysical machinery underlying their electrophysiological properties. These studies have begun to reveal how a diverse array of distinct, cytoplasmically positioned domains affect the activity of associated channels. Some of these establish functional diversity by selectively mediating channel assembly. More importantly, these cytoplasmic domains couple intracellular signals to the gating of their associated pore. New structural insights are providing a clearer understanding of the fundamental molecular mechanisms of these K(+) channels that, in turn, partly underlie complex neurological phenomena.
Asunto(s)
Citoplasma/metabolismo , Canales de Potasio con Entrada de Voltaje/química , Canales de Potasio con Entrada de Voltaje/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Canales de Potasio con Entrada de Voltaje/genética , Estructura Terciaria de ProteínaRESUMEN
BACKGROUND: Mistic is a unique Bacillus subtilis protein with virtually no detectable homologues in GenBank, which appears to integrate into the bacterial membrane despite an overall hydrophilic composition. These unusual properties have been shown to be useful for high-yield recombinant expression of other membrane proteins through fusion to the C-terminus of Mistic. To better understand the structure and function of Mistic, we systematically searched for and characterized homologous proteins among closely related bacteria. RESULTS: Three homologues of Mistic were found with 62% to 93% residue identity, all only 84 residues in length, corresponding to the C-terminal residues of B. subtilis Mistic. In every case, the Mistic gene was found partially overlapping a downstream gene for a K+ channel protein. Residue variation amongst these sequences is restricted to loop regions of the protein's structure, suggesting that secondary structure elements and overall fold have been conserved. Additionally, all three homologues retain the functional ability to chaperone fusion partners to the membrane. CONCLUSION: The functional core of Mistic consists of 84 moderately conserved residues that are sufficient for membrane targeting and integration. Understanding the minimal structural and chemical complexity of Mistic will lead to insights into the mechanistic underpinnings of Mistic-chaperoned membrane integration, as well as how to optimize its use for the recombinant heterologous expression of other integral membrane proteins of interest.
Asunto(s)
Bacillus subtilis/química , Proteínas Bacterianas/química , Secuencia Conservada , Secuencia de Aminoácidos , Aminoácidos/química , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Clonación Molecular , Secuencia Conservada/genética , Genes Bacterianos/genética , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Relación Estructura-ActividadRESUMEN
The inherent difficulties of stabilizing detergent-solubilized integral membrane proteins for biophysical or structural analysis demand the development of new methodologies to improve success rates. One proven strategy is the use of antibody fragments to increase the ;soluble' portion of any membrane protein, but this approach is limited by the difficulties and expense associated with producing monoclonal antibodies to an appropriate exposed epitope on the target protein. Here, the stabilization of a detergent-solubilized K(+) channel protein, KvPae, by engineering a FLAG-binding epitope into a known loop region of the protein and creating a complex with Fab fragments from commercially available anti-FLAG M2 monoclonal antibodies is reported. Although well diffracting crystals of the complex have not yet been obtained, during the course of crystallization trials the structure of the anti-FLAG M2 Fab domain was solved to 1.86 A resolution. This structure, which should aid future structure-determination efforts using this approach by facilitating molecular-replacement phasing, reveals that the binding pocket appears to be specific only for the first four amino acids of the traditional FLAG epitope, namely DYKD. Thus, the use of antibody fragments for improving the stability of target proteins can be rapidly applied to the study of membrane-protein structure by placing the short DKYD motif within a predicted peripheral loop of that protein and utilizing commercially available anti-FLAG M2 antibody fragments.
Asunto(s)
Anticuerpos Monoclonales/química , Fragmentos Fab de Inmunoglobulinas/química , Péptidos/química , Ingeniería de Proteínas/métodos , Secuencia de Aminoácidos , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Sitios de Unión de Anticuerpos/genética , Cristalización , Cristalografía por Rayos X , Epítopos/genética , Epítopos/inmunología , Epítopos/metabolismo , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/metabolismo , Datos de Secuencia Molecular , Oligopéptidos , Péptidos/genética , Péptidos/inmunología , Péptidos/metabolismo , Estructura Terciaria de Proteína , TermodinámicaRESUMEN
Even though the structure determination of soluble proteins has become routine, the number of unrelated integral membrane protein structures remains at a few dozen. The importance of this class of proteins to the molecular mechanisms underlying numerous biological phenomena demands that novel experimental techniques be developed to overcome the limitations imposed by conventional detergent-dependent approaches. Here we report the re-engineering of a putative K+ channel protein of unknown structure into a water-soluble analogue. By analyzing evolutionary conservation patterns of related sequences, lipid-facing residues of the primitive channel were identified and mutagenized into more polar alternatives. Further stabilization of the resultant construct was achieved through fusion with maltose-binding protein. The final soluble protein forms a tetramer, suggesting that it accurately models its predecessor. This methodology, as a viable alternative to the use of detergents, should be applicable to a wide range of integral membrane protein families including transporters and other signal transducers.
Asunto(s)
Proteínas de la Membrana/química , Canales de Potasio/química , Secuencia de Aminoácidos , Guanidina/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Canales de Potasio/genética , Homología de Secuencia de AminoácidoRESUMEN
Uridine phosphorylase (UPP) is a central enzyme in the pyrimidine salvage pathway, catalyzing the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate. Human UPP activity has been a focus of cancer research due to its role in activating fluoropyrimidine nucleoside chemotherapeutic agents such as 5-fluorouracil (5-FU) and capecitabine. Additionally, specific molecular inhibitors of this enzyme have been found to raise endogenous uridine concentrations, which can produce a cytoprotective effect on normal tissues exposed to these drugs. Here we report the structure of hUPP1 bound to 5-FU at 2.3 A resolution. Analysis of this structure reveals new insights as to the conformational motions the enzyme undergoes in the course of substrate binding and catalysis. The dimeric enzyme is capable of a large hinge motion between its two domains, facilitating ligand exchange and explaining observed cooperativity between the two active sites in binding phosphate-bearing substrates. Further, a loop toward the back end of the uracil binding pocket is shown to flexibly adjust to the varying chemistry of different compounds through an "induced-fit" association mechanism that was not observed in earlier hUPP1 structures. The details surrounding these dynamic aspects of hUPP1 structure and function provide unexplored avenues to develop novel inhibitors of this protein with improved specificity and increased affinity. Given the recent emergence of new roles for uridine as a neuron protective compound in ischemia and degenerative diseases, such as Alzheimer's and Parkinson's, inhibitors of hUPP1 with greater efficacy, which are able to boost cellular uridine levels without adverse side-effects, may have a wide range of therapeutic applications.
Asunto(s)
Uridina Fosforilasa/química , Biocatálisis , Dominio Catalítico , Cristalografía por Rayos X , Dimerización , Fluorouracilo/metabolismo , Humanos , Conformación Molecular , Unión Proteica , Conformación Proteica , Especificidad por Sustrato , Uridina/metabolismo , Uridina Fosforilasa/genética , Uridina Fosforilasa/metabolismoRESUMEN
KTN (RCK) domains are nucleotide-binding folds that form the cytoplasmic regulatory complexes of various K+ channels and transporters. The mechanisms these proteins use to control their transmembrane pore-forming counterparts remains unclear despite numerous electrophysiological and structural studies. KTN (RCK) domains consistently crystallize as dimers within the asymmetric unit, forming a pronounced hinge between two Rossmann folds. We have previously proposed that modification of the hinge angle plays an important role in activating the associated membrane-integrated components of the channel or transporter. Here we report the structure of the C-terminal, KTN-bearing domain of the E. coli KefC K+ efflux system in association with the ancillary subunit, KefF, which is known to stabilize the conductive state. The structure of the complex and functional analysis of KefC variants reveal that control of the conformational flexibility inherent in the KTN dimer hinge is modulated by KefF and essential for regulation of KefC ion flux.
Asunto(s)
Membrana Celular/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Canales de Potasio de Rectificación Interna/química , Canales de Potasio de Rectificación Interna/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Secuencia Conservada , Dimerización , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/aislamiento & purificación , Proteínas de Escherichia coli/metabolismo , Secuencias Hélice-Giro-Hélice , Interacciones Hidrofóbicas e Hidrofílicas , Datos de Secuencia Molecular , Mutación , Canales de Potasio/genética , Canales de Potasio/aislamiento & purificación , Canales de Potasio/metabolismo , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Although structure determination of soluble proteins has become routine, our understanding of membrane proteins has been limited by experimental bottlenecks in obtaining both sufficient yields of protein and ordered crystals. Mistic is an unusual Bacillus subtilis integral membrane protein that folds autonomously into the membrane, bypassing the cellular translocon machinery. Using paramagnetic probes, we determined by nuclear magnetic resonance (NMR) spectroscopy that the protein forms a helical bundle with a surprisingly polar lipid-facing surface. Additional experiments suggest that Mistic can be used for high-level production of other membrane proteins in their native conformations, including many eukaryotic proteins that have previously been intractable to bacterial expression.
Asunto(s)
Bacillus subtilis/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Membrana Celular/química , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli , Enlace de Hidrógeno , Membrana Dobles de Lípidos , Micelas , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Mutación , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Receptores de Factores de Crecimiento Transformadores beta/química , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Regulation of intracellular potassium levels is one of the basic functions of all cells, controlling cellular osmolarity and transmitting information. In higher organisms, elaborate control of transmembrane potassium flux has evolved to endow nervous systems with the remarkable ability to transmit electrical signals between cells. Multiple genes, gene splicing, mRNA editing, and selective tetrameric assembly of K channel genes provide the basis for creating distinct electrophysiological properties at varying developmental and cellular stages. This assembly mechanism, primarily governed by the T1 domain, is under the control of intracellular signals. Atomic structures of the isolated T1 domains of Shaker and Shaw subfamilies provided us with valuable structural insights into understanding both channel assembly and functional regulation of the entire channel molecule through conformational changes.
Asunto(s)
Canales de Potasio con Entrada de Voltaje/química , Canales de Potasio con Entrada de Voltaje/ultraestructura , Sitios de Unión , Citoplasma/ultraestructura , Dimerización , Ligandos , Sustancias Macromoleculares , Modelos Moleculares , NAD/química , Canales de Potasio con Entrada de Voltaje/genética , Conformación Proteica , Estructura Secundaria de Proteína , Edición de ARN , Empalme del ARNRESUMEN
The regulation of cation content is critical for cell growth. However, the molecular mechanisms that gate the systems that control K+ movements remain unclear. KTN is a highly conserved cytoplasmic domain present ubiquitously in a variety of prokaryotic and eukaryotic K+ channels and transporters. Here we report crystal structures for two representative KTN domains that reveal a dimeric hinged assembly. Alternative ligands NAD+ and NADH block or vacate, respectively, the hinge region affecting the dimer's conformational flexibility. Conserved, surface-exposed hydrophobic patches that become coplanar upon hinge closure provide an assembly interface for KTN tetramerization. Mutational analysis using the KefC system demonstrates that this domain directly interacts with its respective transmembrane constituent, coupling ligand-mediated KTN conformational changes to the permease's activity.