RESUMEN
Mutation of phenylalanine at position 508 in the cystic fibrosis transmembrane conductance regulator (F508del CFTR) yields a protein unstable at physiological temperatures that is rapidly degraded in the cell. This mutation is present in about 90% of cystic fibrosis patients, hence there is great interest in compounds reversing its instability. We have previously reported the expression of the mutated protein at low temperature and its purification in detergent. Here we describe the use of the protein to screen compounds present in a library of Federal Drug Administration (FDA) - approved drugs and also in a small natural product library. The kinetics of unfolding of F508del CFTR at 37 °C were probed by the increase in solvent-exposed cysteine residues accessible to a fluorescent reporter molecule. This occurred in a bi-exponential manner with a major (≈60%) component of half-life around 5 min and a minor component of around 60 min. The faster kinetics match those observed for loss of channel activity of F508del CFTR in cells at 37 °C. Most compounds tested had no effect on the fluorescence increase, but some were identified that significantly slowed the kinetics. The general properties of these compounds, and any likely mechanisms for inducing stability in purified CFTR are discussed. These experimental data may be useful for artificial intelligence - aided design of CFTR-specific drugs and in the identification of stabilizing additives for membrane proteins (in general).
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística , Bibliotecas de Moléculas Pequeñas , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Humanos , Cinética , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/química , Desplegamiento Proteico , Estabilidad Proteica , Fibrosis Quística/metabolismo , Fibrosis Quística/genética , Fibrosis Quística/tratamiento farmacológico , MutaciónRESUMEN
ABCG2 is an important ATP-binding cassette transporter impacting the absorption and distribution of over 200 chemical toxins and drugs. ABCG2 also reduces the cellular accumulation of diverse chemotherapeutic agents. Acquired somatic mutations in the phylogenetically conserved amino acids of ABCG2 might provide unique insights into its molecular mechanisms of transport. Here, we identify a tumor-derived somatic mutation (Q393K) that occurs in a highly conserved amino acid across mammalian species. This ABCG2 mutant seems incapable of providing ABCG2-mediated drug resistance. This was perplexing because it is localized properly and retained interaction with substrates and nucleotides. Using a conformationally sensitive antibody, we show that this mutant appears "locked" in a non-functional conformation. Structural modeling and molecular dynamics simulations based on ABCG2 cryo-EM structures suggested that the Q393K interacts with the E446 to create a strong salt bridge. The salt bridge is proposed to stabilize the inward-facing conformation, resulting in an impaired transporter that lacks the flexibility to readily change conformation, thereby disrupting the necessary communication between substrate binding and transport.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Neoplasias , Humanos , Animales , Transportadoras de Casetes de Unión a ATP/metabolismo , Mutación , Resistencia a Medicamentos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Resistencia a Antineoplásicos/genética , Mamíferos/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismoRESUMEN
ABCG2 is a medically important ATP-binding cassette transporter with crucial roles in the absorption and distribution of chemically-diverse toxins and drugs, reducing the cellular accumulation of chemotherapeutic drugs to facilitate multidrug resistance in cancer. ABCG2's capacity to transport both hydrophilic and hydrophobic compounds is not well understood. Here we assess the molecular basis for substrate discrimination by the binding pocket. Substitution of a phylogenetically-conserved polar residue, N436, to alanine in the binding pocket of human ABCG2 permits only hydrophobic substrate transport, revealing the unique role of N436 as a discriminator. Molecular dynamics simulations show that this alanine substitution alters the electrostatic potential of the binding pocket favoring hydration of the transport pore. This change affects the contact with substrates and inhibitors, abrogating hydrophilic compound transport while retaining the transport of hydrophobic compounds. The N436 residue is also required for optimal transport inhibition of ABCG2, as many inhibitors are functionally impaired by this ABCG2 mutation. Overall, these findings have biomedical implications, broadly extending our understanding of substrate and inhibitor interactions.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Alanina , Humanos , Electricidad Estática , Inhibición Psicológica , Simulación de Dinámica Molecular , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Proteínas de Neoplasias/genéticaRESUMEN
Cystic fibrosis is a hereditary disease mainly caused by the deletion of the Phe 508 (F508del) of the cystic fibrosis transmembrane conductance regulator (CFTR) protein that is thus withheld in the endoplasmic reticulum and rapidly degraded by the ubiquitin/proteasome system. Cystic fibrosis remains a potentially fatal disease, but it has become treatable as a chronic condition due to some CFTR-rescuing drugs that, when used in combination, increase in their therapeutic effect due to a synergic action. Also, dietary supplementation of natural compounds in combination with approved drugs could represent a promising strategy to further alleviate cystic fibrosis symptoms. On these bases, we screened by in silico drug repositioning 846 small synthetic or natural compounds from the AIFA database to evaluate their capacity to interact with the highly druggable lumacaftor binding site of F508del-CFTR. Among the identified hits, nicotinamide (NAM) was predicted to accommodate into the lumacaftor binding region of F508del-CFTR without competing against the drug but rather stabilizing its binding. The effective capacity of NAM to bind F508del-CFTR in a lumacaftor-uncompetitive manner was then validated experimentally by surface plasmon resonance analysis. Finally, the capacity of NAM to synergize with lumacaftor increasing its CFTR-rescuing activity was demonstrated in cell-based assays. This study suggests the possible identification of natural small molecules devoid of side effects and endowed with the capacity to synergize with drugs currently employed for the treatment of cystic fibrosis, which hopefully will increase the therapeutic efficacy with lower doses.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Humanos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Reposicionamiento de Medicamentos , Complejo de la Endopetidasa Proteasomal/metabolismo , Benzodioxoles/farmacología , Benzodioxoles/uso terapéutico , Aminopiridinas/farmacología , Aminopiridinas/uso terapéutico , Niacinamida/uso terapéutico , Ubiquitinas/metabolismo , MutaciónRESUMEN
ATP-binding cassette (ABC) transporters are one of the largest families of membrane proteins in prokaryotic organisms. Much is now understood about the structure of these transporters and many reviews have been written on that subject. In contrast, less has been written on the assembly of ABC transporter complexes and this will be a major focus of this book chapter. The complexes are formed from two cytoplasmic subunits that are highly conserved (in terms of their primary and three-dimensional structures) across the whole family. These ATP-binding subunits give rise to the name of the family. They must assemble with two transmembrane subunits that will typically form the permease component of the transporter. The transmembrane subunits have been found to be surprisingly diverse in structure when the whole family is examined, with seven distinct folds identified so far. Hence nucleotide-binding subunits appear to have been bolted on to a variety of transmembrane platforms during evolution, leading to a greater variety in function. Furthermore, many importers within the family utilise a further external substrate-binding component to trap scarce substrates and deliver them to the correct permease components. In this chapter, we will discuss whether assembly of the various ABC transporter subunits occurs with high fidelity within the crowded cellular environment and whether promiscuity in assembly of transmembrane and cytoplasmic components can occur. We also discuss the new AlphaFold protein structure prediction tool which predicts a new type of transmembrane domain fold within the ABC transporters that is associated with cation exporters of bacteria and plants.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Proteínas de Transporte de Membrana , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Nucleótidos/metabolismo , Células Procariotas/metabolismoRESUMEN
ABCB1/P-glycoprotein is an ATP binding cassette transporter that is involved in the clearance of xenobiotics, and it affects the disposition of many drugs in the body. Conformational flexibility of the protein within the membrane is an intrinsic part of its mechanism of action, but this has made structural studies challenging. Here, we have studied different conformations of P-glycoprotein simultaneously in the presence of ivacaftor, a known competitive inhibitor. In order to conduct this, we used high contrast cryo-electron microscopy imaging with a Volta phase plate. We associate the presence of ivacaftor with the appearance of an additional density in one of the conformational states detected. The additional density is in the central aqueous cavity and is associated with a wider separation of the two halves of the transporter in the inward-facing state. Conformational changes to the nucleotide-binding domains are also observed and may help to explain the stimulation of ATPase activity that occurs when transported substrate is bound in many ATP binding cassette transporters.
RESUMEN
Anti-viral small molecules are currently lacking for treating coronavirus infection. The long development timescales for such drugs are a major problem, but could be shortened by repurposing existing drugs. We therefore screened a small library of FDA-approved compounds for potential severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antivirals using a pseudovirus system that allows a sensitive read-out of infectivity. A group of structurally-related compounds, showing moderate inhibitory activity with IC50 values in the 2-5 µM range, were identified. Further studies demonstrated that these "kite-shaped" molecules were surprisingly specific for SARS-CoV-1 and SARS-CoV-2 and that they acted early in the entry steps of the viral infectious cycle, but did not affect virus attachment to the cells. Moreover, the compounds were able to prevent infection in both kidney- and lung-derived human cell lines. The structural homology of the hits allowed the production of a well-defined pharmacophore that was found to be highly accurate in predicting the anti-viral activity of the compounds in the screen. We discuss the prospects of repurposing these existing drugs for treating current and future coronavirus outbreaks.
Asunto(s)
Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , COVID-19/virología , Virus de la Leucemia Murina/efectos de los fármacos , SARS-CoV-2 , Internalización del Virus/efectos de los fármacos , Animales , Línea Celular , Chlorocebus aethiops , Descubrimiento de Drogas/métodos , Reposicionamiento de Medicamentos , Sinergismo Farmacológico , Humanos , Virus de la Leucemia Murina/metabolismo , Ratones , Simulación del Acoplamiento Molecular , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células Vero , Acoplamiento Viral/efectos de los fármacosRESUMEN
ATP-binding cassette (ABC) transporters are ubiquitous across all realms of life. Dogma suggests that bacterial ABC transporters include both importers and exporters, whilst eukaryotic members of this family are solely exporters, implying that ABC import function was lost during evolution. This view is being challenged, for example energy-coupling factor (ECF)-type ABC importers appear to fulfil important roles in both algae and plants where they form the ABCI sub-family. Herein we discuss whether bacterial Type I and Type II ABC importers also made the transition into extant eukaryotes. Various studies suggest that Type I importers exist in algae and the liverwort family of primitive non-vascular plants, but not in higher plants. The existence of eukaryotic Type II importers is also supported: a transmembrane protein homologous to vitamin B12 import system transmembrane protein (BtuC), hemin transport system transmembrane protein (HmuU) and high-affinity zinc uptake system membrane protein (ZnuB) is present in the Cyanophora paradoxa genome. This protein has homologs within the genomes of red algae. Furthermore, its candidate nucleotide-binding domain (NBD) shows closest similarity to other bacterial Type II importer NBDs such as BtuD. Functional studies suggest that Type I importers have roles in maintaining sulphate levels in the chloroplast, whilst Type II importers probably act as importers of Mn2+ or Zn2+ , as inferred by comparisons with bacterial homologs. Possible explanations for the presence of these transporters in simple plants, but not in other eukaryotic organisms, are considered. In order to utilise the existing nomenclature for eukaryotic ABC proteins, we propose that eukaryotic Type I and II importers be classified as ABCJ and ABCK transporters, respectively.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Adenosina Trifosfato , Transportadoras de Casetes de Unión a ATP/genética , Eucariontes/genéticaRESUMEN
The emergence of COVID-19 has presented employees and employers new challenges as many employees and managers were forced to work in a remote environment for the first time. For many reasons, managing virtual teams is different than managing employees in a traditional face-to-face office environment. Although many managers have been learning how to lead their virtual teams over the last several months, we offer five steps for leaders to follow for how to maximize the effectiveness of a remote workplace. By taking specific actions and ensuring the organization has a culture to support their virtual workforce, leaders can improve the performance output and engagement of their teams. The five steps are: first establish and explain the new reality; second, establish and maintain a culture of trust; third, upgrade leadership communication tools and techniques to better inform virtual employees; fourth, encourage shared leadership among team members; and fifth, to create and periodically perform alignment audits to ensure virtual employees are aligned with the organization's cultural values including its commitment to mission. All these steps start with the realization that managing a team is going to be different when the members are dispersed, and new leadership strategies, communication routines and tools are required.
RESUMEN
The classic conceptualization of ATP binding cassette (ABC) transporter function is an ATP-dependent conformational change coupled to transport of a substrate across a biological membrane via the transmembrane domains (TMDs). The binding of two ATP molecules within the transporter's two nucleotide binding domains (NBDs) induces their dimerization. Despite retaining the ability to bind nucleotides, isolated NBDs frequently fail to dimerize. ABC proteins without a TMD, for example ABCE and ABCF, have NBDs tethered via elaborate linkers, further supporting that NBD dimerization does not readily occur for isolated NBDs. Intriguingly, even in full-length transporters, the NBD-dimerized, outward-facing state is not as frequently observed as might be expected. This leads to questions regarding what drives NBD interaction and the role of the TMDs or linkers. Understanding the NBD-nucleotide interaction and the subsequent NBD dimerization is thus pivotal for understanding ABC transporter activity in general. Here, we hope to provide new insights into ABC protein function by discussing the perplexing issue of (missing) NBD dimerization in isolation and in the context of full-length ABC proteins.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Dominios Proteicos , Multimerización de Proteína , Animales , Sitios de Unión , Humanos , Unión ProteicaRESUMEN
Cystic fibrosis transmembrane conductance regulator (CFTR)-rescuing drugs have already transformed cystic fibrosis (CF) from a fatal disease to a treatable chronic condition. However, new-generation drugs able to bind CFTR with higher specificity/affinity and to exert stronger therapeutic benefits and fewer side effects are still awaited. Computational methods and biosensors have become indispensable tools in the process of drug discovery for many important human pathologies. Instead, they have been used only piecemeal in CF so far, calling for their appropriate integration with well-tried CF biochemical and cell-based models to speed up the discovery of new CFTR-rescuing drugs. This review will give an overview of the available structures and computational models of CFTR and of the biosensors, biochemical and cell-based assays already used in CF-oriented studies. It will also give the reader some insights about how to integrate these tools as to improve the efficiency of the drug discovery process targeted to CFTR.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/efectos de los fármacos , Descubrimiento de Drogas/métodos , Técnicas Biosensibles , Biología Computacional , Fibrosis Quística/tratamiento farmacológico , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Humanos , Modelos Moleculares , Conformación ProteicaRESUMEN
ATP-binding cassette sub-family G member 2 (ABCG2) is a homodimeric ATP-binding cassette (ABC) transporter that not only has a key role in helping cancer cells to evade the cytotoxic effects of chemotherapy, but also in protecting organisms from multiple xeno- and endobiotics. Structural studies indicate that substrate and inhibitor (ligands) binding to ABCG2 can be differentiated quantitatively by the number of amino acid contacts, with inhibitors displaying more contacts. Although binding is the obligate initial step in the transport cycle, there is no empirical evidence for one amino acid being primarily responsible for ligand binding. By mutagenesis and biochemical studies, we demonstrated that the phylogenetically conserved amino acid residue, F439, was critical for both transport and the binding of multiple substrates and inhibitors. Structural modeling implied that the π-π interactions from each F439 monomer mediated the binding of a surprisingly diverse array of structurally unrelated substrates and inhibitors and that this symmetrical π-π interaction "clamps" the ligand into the binding pocket. Key molecular features of diverse ABCG2 ligands using the π-π clamp along with structural studies created a pharmacophore model. These novel findings have important therapeutic implications because key properties of ligands interacting with ABCG2 have been disovered. Furthermore, mechanistic insights have been revealed by demonstrating that for ABCG2 a single amino acid is essential for engaging and initiating transport of multiple drugs and xenobiotics.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/química , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/antagonistas & inhibidores , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Animales , Sitios de Unión , Células Cultivadas , Células HEK293 , Humanos , Lapatinib/análogos & derivados , Lapatinib/farmacología , Ratones , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Crystallization of recombinant proteins has been fundamental to our understanding of protein function, dysfunction, and molecular recognition. However, this information has often been gleaned under extremely nonphysiological protein, salt, and H+ concentrations. Here, we describe the development of a robust Inka1-Box (iBox)-PAK4cat system that spontaneously crystallizes in several mammalian cell types. The semi-quantitative assay described here allows the measurement of in vivo protein-protein interactions using a novel GFP-linked reporter system that produces fluorescent readouts from protein crystals. We combined this assay with in vitro X-ray crystallography and molecular dynamics studies to characterize the molecular determinants of the interaction between the PDZ2 domain of Na+/H+ exchange regulatory cofactor NHE-RF1 (NHERF1) and cystic fibrosis transmembrane conductance regulator (CFTR), a protein complex pertinent to the genetic disease cystic fibrosis. These experiments revealed the crystal structure of the extended PDZ domain of NHERF1 and indicated, contrary to what has been previously reported, that residue selection at positions -1 and -3 of the PDZ-binding motif influences the affinity and specificity of the NHERF1 PDZ2-CFTR interaction. Our results suggest that this system could be utilized to screen additional protein-protein interactions, provided they can be accommodated within the spacious iBox-PAK4cat lattice.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fosfoproteínas/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS , Chlorocebus aethiops , Cristalografía por Rayos X , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Humanos , Simulación de Dinámica Molecular , Dominios PDZ , Fosfoproteínas/química , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Alineación de Secuencia , Intercambiadores de Sodio-Hidrógeno/química , TermodinámicaRESUMEN
ATP-binding cassette (ABC) transporters are membrane proteins present in all kingdoms of life. We have considered the disordered region that connects the N- and C-terminal halves in many eukaryotic ABC transporters, allowing all four consensus functional domains to be linked. The recent availability of structures of ABC transporters containing linker regions has allowed us to identify the start and end points of the connectors as well as hinting at their localisation. We address questions such as: Where did the linker regions come from? Why do some ABC transporters have connectors and others not? What are the rules and roles of the linker regions? What are the consequences of mutations in these connector regions for disease in humans?
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Secuencia de Aminoácidos , Humanos , Mutación , Procesamiento Proteico-Postraduccional , Relación Estructura-ActividadRESUMEN
Cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ATP-binding cassette family of proteins because it has evolved into a channel. Mutations in CFTR cause cystic fibrosis, the most common genetic disease in people of European origin. The F508del mutation is found in about 90% of patients and here we present data that suggest its main effect is on CFTR stability rather than on the three-dimensional (3D) folded state. A survey of recent cryo-electron microscopy studies was carried out and this highlighted differences in terms of CFTR conformation despite similarities in experimental conditions. We further studied CFTR structure under various phosphorylation states and with the CFTR-interacting protein NHERF1. The coexistence of outward-facing and inward-facing conformations under a range of experimental conditions was suggested from these data. These results are discussed in terms of structural models for channel gating, and favour the model where the mostly disordered regulatory-region of the protein acts as a channel plug.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Microscopía por Crioelectrón , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Humanos , Mutación , Fosfoproteínas/metabolismo , Fosforilación , Conformación Proteica , Estabilidad Proteica , Proteolisis , Intercambiadores de Sodio-Hidrógeno/metabolismoRESUMEN
ATP-binding cassette (ABC) transporters are essential proteins that are found across all kingdoms of life. ABC transporters harness the energy of ATP hydrolysis to drive the import of nutrients inside bacterial cells or the export of toxic compounds or essential lipids across bacteria and eukaryotic membranes. Typically, ABC transporters consist of transmembrane domains (TMDs) and nucleotide-binding domains (NBDs) to bind their substrate and ATP, respectively. The TMDs dictate what ligands can be recognised, whereas the NBDs are the power engine of the ABC transporter, carrying out ATP binding and hydrolysis. It has been proposed that they utilise the alternating access mechanism, inward- to outward-facing conformation, to transport their substrates. Here, we will review the recent progress on the structure determination of eukaryotic and bacterial ABC transporters as well as the novel mechanisms that have also been proposed, that fall out of the alternating access mechanism model.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Animales , Humanos , Simulación de Dinámica Molecular , Unión Proteica , Conformación ProteicaRESUMEN
BACKGROUND: P-glycoprotein (ABCB1) is an ATP-binding cassette transporter that plays an important role in the clearance of drugs and xenobiotics and is associated with multi-drug resistance in cancer. Although several P-glycoprotein structures are available, these are either at low resolution, or represent mutated and/or quiescent states of the protein. RESULTS: In the post-hydrolytic state the structure of the wild-type protein has been resolved at about 8 Å resolution. The cytosolic nucleotide-binding domains (NBDs) are separated but ADP remains bound, especially at the first NBD. Gaps in the transmembrane domains (TMDs) that connect to an inner hydrophilic cavity are filled by density emerging from the annular detergent micelle. The NBD-TMD linker is partly resolved, being located between the NBDs and close to the Signature regions involved in cooperative NBD dimerization. This, and the gap-filling detergent suggest steric impediment to NBD dimerization in the post-hydrolytic state. Two central regions of density lie in two predicted drug-binding sites, implying that the protein may adventitiously bind hydrophobic substances even in the post-hydrolytic state. The previously unresolved N-terminal extension was observed, and the data suggests these 30 residues interact with the headgroup region of the lipid bilayer. CONCLUSION: The structural data imply that (i) a low basal ATPase activity is ensured by steric blockers of NBD dimerization and (ii) allocrite access to the central cavity may be structurally linked to NBD dimerization, giving insights into the mechanism of drug-stimulation of P-glycoprotein activity.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Microscopía por Crioelectrón , Humanos , Ratones , Péptidos/química , Péptidos/metabolismo , Conformación Proteica , Dominios Proteicos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
CFTR (ABCC7) is a phospho-regulated chloride channel that is found in the apical membranes of epithelial cells, is gated by ATP and the activity of the protein is crucial in the homeostasis of the extracellular liquid layer in many organs [Annu. Rev. Biochem. (2008) 77, 701-726; Science (1989) 245, 1066-1073]. Mutations in CFTR cause the inherited disease cystic fibrosis (CF), the most common inherited condition in humans of European descent [Science (1989) 245, 1066-1073; Pflugers Arch. (2007) 453, 555-567]. The structural basis of CF will be discussed in this article.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Adenosina Trifosfato/química , Animales , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células Epiteliales/metabolismo , Homeostasis , Humanos , Activación del Canal Iónico , Mutación , Fosforilación , Dominios Proteicos , Relación Estructura-Actividad , Pez CebraRESUMEN
Membrane proteins represent a large proportion of the proteome, but have characteristics that are problematic for many methods in modern molecular biology (that have often been developed with soluble proteins in mind). For structural studies, low levels of expression and the presence of detergent have been thorns in the flesh of the membrane protein experimentalist. Here we discuss the use of cryo-electron microscopy in breakthrough studies of the structures of membrane proteins. This method can cope with relatively small quantities of sample and with the presence of detergent. Until recently, cryo-electron microscopy could not deliver high-resolution structures of membrane proteins, but recent developments in transmission electron microscope technology and in the image processing of single particles imaged in the microscope have revolutionized the field, allowing high resolution structures to be obtained. Here we focus on the specific issues surrounding the application of cryo-electron microscopy to the study of membrane proteins, especially in the choice of a system to keep the protein soluble.
Asunto(s)
Microscopía por Crioelectrón/métodos , Proteínas de la Membrana/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Humanos , Maleatos/química , Micelas , Poliestirenos/químicaRESUMEN
Ivacaftor is a potentiator of the CFTR chloride channel and is in worldwide clinical use for the chronic treatment of cystic fibrosis in patients. There is evidence that the bioavailability of ivacaftor in the body may be influenced by the multi-drug exporter P-glycoprotein. Here we have employed purified and reconstituted P-glycoprotein to study its interaction with ivacaftor as well as the ability of the drug to compete with a known transported substrate of the protein. We find that ivacaftor stimulates the ATPase activity of the purified protein and can compete with the transport of the fluorescent substrate Hoechst 33342. These findings lead us to conclude that ivacaftor is very likely an efficiently transported substrate of P-glycoprotein. Evidence for state-dependent binding of ivacaftor was obtained using a fluorescent, cysteine-reactive reporter dye. The quiescent, nucleotide-free state in the P-glycoprotein transport cycle appears to bind ivacaftor strongly.