RESUMEN
Eukaryotic Ca(2+) regulation involves sequestration into intracellular organelles, and expeditious Ca(2+) release into the cytosol is a hallmark of key signalling transduction pathways. Bulk removal of Ca(2+) after such signalling events is accomplished by members of the Ca(2+):cation (CaCA) superfamily. The CaCA superfamily includes the Na(+)/Ca(2+) (NCX) and Ca(2+)/H(+) (CAX) antiporters, and in mammals the NCX and related proteins constitute families SLC8 and SLC24, and are responsible for the re-establishment of Ca(2+) resting potential in muscle cells, neuronal signalling and Ca(2+) reabsorption in the kidney. The CAX family members maintain cytosolic Ca(2+) homeostasis in plants and fungi during steep rises in intracellular Ca(2+) due to environmental changes, or following signal transduction caused by events such as hyperosmotic shock, hormone response and response to mating pheromones. The cytosol-facing conformations within the CaCA superfamily are unknown, and the transport mechanism remains speculative. Here we determine a crystal structure of the Saccharomyces cerevisiae vacuolar Ca(2+)/H(+) exchanger (Vcx1) at 2.3 Å resolution in a cytosol-facing, substrate-bound conformation. Vcx1 is the first structure, to our knowledge, within the CAX family, and it describes the key cytosol-facing conformation of the CaCA superfamily, providing the structural basis for a novel alternating access mechanism by which the CaCA superfamily performs high-throughput Ca(2+) transport across membranes.
Asunto(s)
Antiportadores/química , Antiportadores/metabolismo , Calcio/metabolismo , Citosol/metabolismo , Protones , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/química , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Transporte Iónico , Methanococcus/química , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Pliegue de Proteína , Relación Estructura-ActividadRESUMEN
Phosphate is crucial for structural and metabolic needs, including nucleotide and lipid synthesis, signalling and chemical energy storage. Proton-coupled transporters of the major facilitator superfamily (MFS) are essential for phosphate uptake in plants and fungi, and also have a function in sensing external phosphate levels as transceptors. Here we report the 2.9 Å structure of a fungal (Piriformospora indica) high-affinity phosphate transporter, PiPT, in an inward-facing occluded state, with bound phosphate visible in the membrane-buried binding site. The structure indicates both proton and phosphate exit pathways and suggests a modified asymmetrical 'rocker-switch' mechanism of phosphate transport. PiPT is related to several human transporter families, most notably the organic cation and anion transporters of the solute carrier family (SLC22), which are implicated in cancer-drug resistance. We modelled representative cation and anion SLC22 transporters based on the PiPT structure to surmise the structural basis for substrate binding and charge selectivity in this important family. The PiPT structure demonstrates and expands on principles of substrate transport by the MFS transporters and illuminates principles of phosphate uptake in particular.
Asunto(s)
Basidiomycota/química , Células Eucariotas/química , Proteínas de Transporte de Fosfato/química , Sitios de Unión , Cristalografía por Rayos X , Humanos , Modelos Biológicos , Modelos Moleculares , Proteínas de Transporte de Fosfato/metabolismo , Fosfatos/metabolismo , Conformación Proteica , Protones , Relación Estructura-ActividadRESUMEN
Nucleosides play an essential role in the physiology of eukaryotes by acting as metabolic precursors in de novo nucleic acid synthesis and energy metabolism. Nucleosides also act as ligands for purinergic receptors. Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that aid in regulating plasmalemmal flux of purine and pyrimidine nucleosides and nucleobases. ENTs exhibit broad substrate selectivity across different isoforms and utilize diverse mechanisms to drive substrate flux across membranes. However, the molecular mechanisms and chemical determinants of ENT-mediated substrate recognition, binding, inhibition, and transport are poorly understood. To determine how ENT-mediated transport occurs at the molecular level, greater chemical insight and assays employing purified protein are essential. This article focuses on the expression and purification of human ENT1, human ENT2, and Saccharomyces cerevisiae ScENT1 using novel expression and purification strategies to isolate recombinant ENTs. ScENT1, hENT1, and hENT2 were expressed in W303 Saccharomyces cerevisiae cells and detergent solubilized from the membrane. After detergent extraction, these ENTs were further purified using immobilized metal affinity chromatography and size exclusion chromatography. This effort resulted in obtaining quantities of purified protein sufficient for future biophysical analysis.
Asunto(s)
Tranportador Equilibrativo 1 de Nucleósido/genética , Transportador Equilibrativo 2 de Nucleósido/genética , Plásmidos/química , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/genética , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Cromatografía de Afinidad , Cromatografía en Gel , Clonación Molecular , Detergentes/química , Tranportador Equilibrativo 1 de Nucleósido/biosíntesis , Tranportador Equilibrativo 1 de Nucleósido/aislamiento & purificación , Transportador Equilibrativo 2 de Nucleósido/biosíntesis , Transportador Equilibrativo 2 de Nucleósido/aislamiento & purificación , Expresión Génica , Humanos , Plásmidos/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Proteínas de Transporte Vesicular/biosíntesis , Proteínas de Transporte Vesicular/aislamiento & purificaciónRESUMEN
Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that transport nucleosides and, to a lesser extent, nucleobases across cell membranes. ENTs modulate efficacy for a range of human therapeutics and function in a diffusion-controlled bidirectional manner. A detailed understanding of ENT function at the molecular level has remained elusive. FUN26 (function unknown now 26) is a putative ENT homolog from S. cerevisiae that is expressed in vacuole membranes. In the present system, proteoliposome studies of purified FUN26 demonstrate robust nucleoside and nucleobase uptake into the luminal volume for a broad range of substrates. This transport activity is sensitive to nucleoside modifications in the C(2')- and C(5')-positions on the ribose sugar and is not stimulated by a membrane pH differential. [(3)H]Adenine nucleobase transport efficiency is increased â¼4-fold relative to nucleosides tested with no observed [(3)H]adenosine or [(3)H]UTP transport. FUN26 mutational studies identified residues that disrupt (G463A or G216A) or modulate (F249I or L390A) transporter function. These results demonstrate that FUN26 has a unique substrate transport profile relative to known ENT family members and that a purified ENT can be reconstituted in proteoliposomes for functional characterization in a defined system.
Asunto(s)
Proteínas de Transporte de Nucleósido Equilibrativas/metabolismo , Proteínas de Transporte de Membrana/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/metabolismo , Secuencia de Bases , Cartilla de ADN , Cinética , Proteínas de Transporte de Membrana/metabolismo , Reacción en Cadena de la Polimerasa , Proteolípidos , Proteínas de Saccharomyces cerevisiae/metabolismo , SolubilidadRESUMEN
Persistent hurdles impede the successful determination of high-resolution crystal structures of eukaryotic integral membrane proteins (IMP). We designed a high-throughput structural genomics oriented pipeline that seeks to minimize effort in uncovering high-quality, responsive non-redundant targets for crystallization. This "discovery-oriented" pipeline sidesteps two significant bottlenecks in the IMP structure determination pipeline: expression and membrane extraction with detergent. In addition, proteins that enter the pipeline are then rapidly vetted by their presence in the included volume on a size-exclusion column--a hallmark of well-behaved IMP targets. A screen of 384 rationally selected eukaryotic IMPs in baker's yeast Saccharomyces cerevisiae is outlined to demonstrate the results expected when applying this discovery-oriented pipeline to whole-organism membrane proteomes.
Asunto(s)
Proteínas de la Membrana/química , Animales , Cristalografía por Rayos X , Bases de Datos de Proteínas , Genómica , Humanos , Conformación Proteica , Proteoma/análisis , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Membrane proteins serve as cellular gatekeepers, regulators, and sensors. Prior studies have explored the functional breadth and evolution of proteins and families of particular interest, such as the diversity of transport-associated membrane protein families in prokaryotes and eukaryotes, the composition of integral membrane proteins, and family classification of all human G-protein coupled receptors. However, a comprehensive analysis of the content and evolutionary associations between membrane proteins and families in a diverse set of genomes is lacking. Here, a membrane protein annotation pipeline was developed to define the integral membrane genome and associations between 21,379 proteins from 34 genomes; most, but not all of these proteins belong to 598 defined families. The pipeline was used to provide target input for a structural genomics project that successfully cloned, expressed, and purified 61 of our first 96 selected targets in yeast. Furthermore, the methodology was applied (1) to explore the evolutionary history of the substrate-binding transmembrane domains of the human ABC transporter superfamily, (2) to identify the multidrug resistance-associated membrane proteins in whole genomes, and (3) to identify putative new membrane protein families.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Resistencia a Múltiples Medicamentos/genética , Evolución Molecular , Genoma Humano/genética , Proteínas de la Membrana/genética , Receptores Acoplados a Proteínas G/genética , Animales , Genómica/métodos , Humanos , Estructura Secundaria de Proteína/genéticaRESUMEN
4-1BB (CD137, TNFRSF9) is an inducible costimulatory receptor expressed on activated T cells. Clinical trials of two agonist antibodies, utomilumab (PF-05082566) and urelumab (BMS-663513), are ongoing in multiple cancer indications, and both antibodies demonstrate distinct activities in the clinic. To understand these differences, we solved structures of the human 4-1BB/4-1BBL complex, the 4-1BBL trimer alone, and 4-1BB bound to utomilumab or urelumab. The 4-1BB/4-1BBL complex displays a unique interaction between receptor and ligand when compared with other TNF family members. Furthermore, our ligand-only structure differs from previously published data. Utomilumab, a ligand-blocking antibody, binds 4-1BB between CRDs 3 and 4. In contrast, urelumab binds 4-1BB CRD-1, away from the ligand binding site. Finally, cell-based assays demonstrate utomilumab is a milder agonist than urelumab. Collectively, our data provide a deeper understanding of the 4-1BB signaling complex, providing a template for future development of next generation 4-1BB targeted biologics.
Asunto(s)
Ligando 4-1BB/química , Ligando 4-1BB/metabolismo , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Inmunoglobulina G/química , Inmunoglobulina G/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/química , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Anticuerpos Monoclonales Humanizados , Sitios de Unión , Células HEK293 , Humanos , Células Jurkat , Modelos Moleculares , Dominios ProteicosRESUMEN
The commercial success of bispecific antibodies generally has been hindered by the complexities associated with generating appropriate molecules for both research scale and large scale manufacturing purposes. Bispecific IgG (BsIgG) based on two antibodies that use an identical common light chain can be combined with a minimal set of Fc mutations to drive heavy chain heterodimerization in order to address these challenges. However, the facile generation of common light chain antibodies with properties similar to traditional monoclonal antibodies has not been demonstrated and they have only been used sparingly. Here, we describe the design of a synthetic human antibody library based on common light chains to generate antibodies with biochemical and biophysical properties that are indistinguishable to traditional therapeutic monoclonal antibodies. We used this library to generate diverse panels of well-behaved, high affinity antibodies toward a variety of epitopes across multiple antigens, including mouse 4-1BB, a therapeutically important T cell costimulatory receptor. Over 200 BsIgG toward 4-1BB were generated using an automated purification method we developed that enables milligram-scale production of BsIgG. This approach allowed us to identify antibodies with a wide range of agonistic activity that are being used to further investigate the therapeutic potential of antibodies targeting one or more epitopes of 4-1BB.
Asunto(s)
Anticuerpos Biespecíficos , Cadenas Ligeras de Inmunoglobulina , Biblioteca de Péptidos , Ingeniería de Proteínas/métodos , Animales , Humanos , RatonesRESUMEN
This unit describes rapid and generally applicable methods to identify conditions that stabilize membrane proteins using temperature-based denaturation measurements as a proxy for target time-dependent stability. Recent developments with thiol-reactive dyes sensitive to the unmasking of cysteine residues upon protein unfolding have allowed for routine application of thermostability assays to systematically evaluate the stability of membrane protein preparations after various purification procedures. Test conditions can include different lipid cocktails, lipid-detergent micelles, pH, salts, osmolytes, and potential active-site ligands. Identification and use of conditions that stabilize the structure have proven successful in enabling the structure determination of numerous families of membrane proteins that otherwise were intractable.
Asunto(s)
Lípidos/química , Proteínas de la Membrana/química , Proteínas de la Membrana/aislamiento & purificación , Micelas , Cristalografía por Rayos X/métodos , Concentración de Iones de HidrógenoRESUMEN
The budding yeast Saccharomyces cerevisiae is a viable system for the overexpression and functional analysis of eukaryotic integral membrane proteins (IMPs). In this chapter we describe a general protocol for the initial cloning, transformation, overexpression, and subsequent purification of a putative IMP and discuss critical optimization steps and approaches. Since expression and purification are often the two predominant hurdles one will face in studying this difficult class of biological macromolecules the intent is to outline the general workflow while providing insights based upon our collective experience. These insights should facilitate tailoring of the outlined protocol to individual IMPs and expression or purification routines.
Asunto(s)
Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de la Membrana/genética , Saccharomyces cerevisiae/genética , Transformación GenéticaRESUMEN
A medium-throughput approach is used to rapidly identify membrane proteins from a eukaryotic organism that are most amenable to expression in amounts and quality adequate to support structure determination. The goal was to expand knowledge of new membrane protein structures based on proteome-wide coverage. In the first phase, membrane proteins from the budding yeast Saccharomyces cerevisiae were selected for homologous expression in S. cerevisiae, a system that can be adapted to expression of membrane proteins from other eukaryotes. We performed medium-scale expression and solubilization tests on 351 rationally selected membrane proteins from S. cerevisiae. These targets are inclusive of all annotated and unannotated membrane protein families within the organism's membrane proteome. Two hundred seventy-two targets were expressed, and of these, 234 solubilized in the detergent n-dodecyl-beta-D-maltopyranoside. Furthermore, we report the identity of a subset of targets that were purified to homogeneity to facilitate structure determinations. The extensibility of this approach is demonstrated with the expression of 10 human integral membrane proteins from the solute carrier superfamily. This discovery-oriented pipeline provides an efficient way to select proteins from particular membrane protein classes, families, or organisms that may be more suited to structure analysis than others.