RESUMO
Cryo-electron microscopy (cryo-EM) has the capacity to capture molecular machines in action1-3. ATP-binding cassette (ABC) exporters are highly dynamic membrane proteins that extrude a wide range of substances from the cytosol4-6 and thereby contribute to essential cellular processes, adaptive immunity and multidrug resistance7,8. Despite their importance, the coupling of nucleotide binding, hydrolysis and release to the conformational dynamics of these proteins remains poorly resolved, especially for heterodimeric and/or asymmetric ABC exporters that are abundant in humans. Here we present eight high-resolution cryo-EM structures that delineate the full functional cycle of an asymmetric ABC exporter in a lipid environment. Cryo-EM analysis under active turnover conditions reveals distinct inward-facing (IF) conformations-one of them with a bound peptide substrate-and previously undescribed asymmetric post-hydrolysis states with dimerized nucleotide-binding domains and a closed extracellular gate. By decreasing the rate of ATP hydrolysis, we could capture an outward-facing (OF) open conformation-an otherwise transient state vulnerable to substrate re-entry. The ATP-bound pre-hydrolysis and vanadate-trapped states are conformationally equivalent; both comprise co-existing OF conformations with open and closed extracellular gates. By contrast, the post-hydrolysis states from the turnover experiment exhibit asymmetric ATP and ADP occlusion after phosphate release from the canonical site and display a progressive separation of the nucleotide-binding domains and unlocking of the intracellular gate. Our findings reveal that phosphate release, not ATP hydrolysis, triggers the return of the exporter to the IF conformation. By mapping the conformational landscape during active turnover, aided by mutational and chemical modulation of kinetic rates to trap the key intermediates, we resolved fundamental steps of the substrate translocation cycle of asymmetric ABC transporters.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Microscopia Crioeletrônica , Thermus thermophilus/química , Transportadores de Cassetes de Ligação de ATP/ultraestrutura , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Hidrólise , Cinética , Modelos Moleculares , Mutação , Conformação Proteica , Multimerização Proteica , Especificidade por Substrato , Thermus thermophilus/ultraestrutura , Vanadatos/metabolismoRESUMO
Solute carrier family 26 (SLC26) is a family of functionally diverse anion transporters found in all kingdoms of life. Anions transported by SLC26 proteins include chloride, bicarbonate, and sulfate, but also small organic dicarboxylates such as fumarate and oxalate. The human genome encodes ten functional homologs, several of which are causally associated with severe human diseases, highlighting their physiological importance. Here, we review novel insights into the structure and function of SLC26 proteins and summarize the physiological relevance of human members.
Assuntos
Proteínas de Transporte de Ânions , Humanos , Transportadores de Sulfato/metabolismo , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/química , Proteínas de Transporte de Ânions/metabolismo , Ânions/metabolismo , Transporte BiológicoRESUMO
Substrate-binding proteins (SBPs) are part of solute transport systems and serve to increase substrate affinity and uptake rates. In contrast to primary transport systems, the mechanism of SBP-dependent secondary transport is not well understood. Functional studies have thus far focused on Na+-coupled Tripartite ATP-independent periplasmic (TRAP) transporters for sialic acid. Herein, we report the in vitro functional characterization of TAXIPm-PQM from the human pathogen Proteus mirabilis. TAXIPm-PQM belongs to a TRAP-subfamily using a different type of SBP, designated TRAP-associated extracytoplasmic immunogenic (TAXI) protein. TAXIPm-PQM catalyzes proton-dependent α-ketoglutarate symport and its SBP is an essential component of the transport mechanism. Importantly, TAXIPm-PQM represents the first functionally characterized SBP-dependent secondary transporter that does not rely on a soluble SBP, but uses a membrane-anchored SBP instead.
Assuntos
Proteínas de Transporte , Proteínas de Membrana , Humanos , Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Transporte BiológicoRESUMO
By reporting the molar abundance of proteins, absolute quantification determines their stoichiometry in complexes, pathways, or networks. Typically, absolute quantification relies either on protein-specific isotopically labeled peptide standards or on a semiempirical calibration against the average abundance of peptides chosen from arbitrarily selected proteins. In contrast, a generic protein standard FUGIS (fully unlabeled generic internal standard) requires no isotopic labeling, chemical synthesis, or external calibration and is applicable to quantifying proteins of any organismal origin. The median intensity of the peptide peaks produced by the tryptic digestion of FUGIS is used as a single-point calibrant to determine the molar abundance of any codigested protein. Powered by FUGIS, median-based absolute quantification (MBAQ) outperformed other methods of untargeted proteome-wide absolute quantification.
Assuntos
Peptídeos , Proteoma , Calibragem , Marcação por Isótopo/métodos , Peptídeos/química , Padrões de ReferênciaRESUMO
Absolute (molar) quantification of clinically relevant proteins determines their reference values in liquid and solid biopsies. The FastCAT (for Fast-track QconCAT) method employs multiple short (<50 kDa), stable-isotope labeled chimeric proteins (CPs) composed of concatenated quantotypic (Q)-peptides representing the quantified proteins. Each CP also comprises scrambled sequences of reference (R)-peptides that relate its abundance to a single protein standard (bovine serum albumin, BSA). FastCAT not only alleviates the need to purify CP or use sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) but also improves the accuracy, precision, and dynamic range of the absolute quantification by grouping Q-peptides according to the expected abundance of the target proteins. We benchmarked FastCAT against the reference method of MS Western and tested it in the direct molar quantification of neurological markers in human cerebrospinal fluid at the low ng/mL level.
Assuntos
Proteínas , Proteômica , Eletroforese em Gel de Poliacrilamida , Humanos , Peptídeos/metabolismo , Proteômica/métodos , Padrões de ReferênciaRESUMO
The activity of enzymes is subject to regulation at multiple levels. Cooperativity, the interconnected behavior of active sites within a protein complex, directly affects protein activity. Cooperativity is a mode of regulation that requires neither extrinsic factors nor protein modifications. Instead, it allows enzymes themselves to modulate reaction rates. Cooperativity is an important regulatory mechanism in soluble proteins, but also examples of cooperative membrane proteins have been described. In this review, we summarize the current knowledge on interprotomer cooperativity in elevator-type proteins, a class of membrane transporters characterized by large rigid-body movements perpendicular to the membrane, and highlight well-studied examples and experimental approaches.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Bactérias/metabolismo , Sítios de Ligação , Domínio Catalítico , Escherichia coli/metabolismo , Humanos , Cinética , Ligantes , Proteínas de Membrana Transportadoras/metabolismo , Ligação Proteica , Multimerização Proteica , SolubilidadeRESUMO
The uptake of glutamate by synaptic vesicles is mediated by vesicular glutamate transporters (VGLUTs). The central role of these transporters in excitatory neurotransmission underpins their importance as pharmacological targets. Although several compounds inhibit VGLUTs, highly specific inhibitors were so far unavailable, thus limiting applications to in vitro experiments. Besides their potential in pharmacology, specific inhibitors would also be beneficial for the elucidation of transport mechanisms. To overcome this shortage, we generated nanobodies (Nbs) by immunization of a llama with purified rat VGLUT1 and subsequent selection of binders from a phage display library. All identified Nbs recognize cytosolic epitopes, and two of the binders greatly reduced the rate of uptake of glutamate by reconstituted liposomes and subcellular fractions enriched with synaptic vesicles. These Nbs can be expressed as functional green fluorescent protein fusion proteins in the cytosol of HEK cells for intracellular applications as immunocytochemical and biochemical agents. The selected binders thus provide valuable tools for cell biology and neuroscience.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Córtex Cerebral/efeitos dos fármacos , Moduladores de Transporte de Membrana/farmacologia , Modelos Moleculares , Proteínas do Tecido Nervoso/antagonistas & inibidores , Neurônios/efeitos dos fármacos , Anticorpos de Domínio Único/farmacologia , Proteína Vesicular 1 de Transporte de Glutamato/antagonistas & inibidores , Animais , Transporte Biológico/efeitos dos fármacos , Camelídeos Americanos , Células Cultivadas , Depressores do Sistema Nervoso Central/química , Depressores do Sistema Nervoso Central/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Embrião de Mamíferos/citologia , Ácido Glutâmico/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Moduladores de Transporte de Membrana/química , Moduladores de Transporte de Membrana/metabolismo , Camundongos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Biblioteca de Peptídeos , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/metabolismo , Proteína Vesicular 1 de Transporte de Glutamato/química , Proteína Vesicular 1 de Transporte de Glutamato/genética , Proteína Vesicular 1 de Transporte de Glutamato/metabolismoRESUMO
Solute carriers from the SLC4, SLC23, and SLC26 families are involved in pH regulation, vitamin C transport and ion homeostasis. While these families do not share any obvious sequence relationship, they are united by their unique and novel architecture. Each member of this structural class is organized into two structurally related halves of seven transmembrane segments each. These halves span the membrane with opposite orientations and form an intricately intertwined structure of two inverted repeats. This review highlights the general design principles of this fold and reveals the diversity between the different families. We discuss their domain architecture, structural framework and transport mode and detail an initial transport mechanism for this fold inferred from the recently solved structures of different members.
Assuntos
Membrana Celular/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Sequências Repetitivas de Aminoácidos , Animais , Transporte Biológico , Humanos , Domínios Proteicos , Multimerização ProteicaRESUMO
ABC exporters are ubiquitous multidomain transport proteins that couple ATP hydrolysis at a pair of nucleotide binding domains to substrate transport across the lipid bilayer mediated by two transmembrane domains. Recently, the crystal structure of the heterodimeric ABC exporter TM287/288 was determined. One of its asymmetric ATP binding sites is called the degenerate site; it binds nucleotides tightly but is impaired in terms of ATP hydrolysis. Here we report the crystal structures of both isolated motor domains of TM287/288. Unexpectedly, structural elements constituting the degenerate ATP binding site are disordered in these crystals and become structured only in the context of the full-length transporter. In addition, hydrogen bonding patterns of key residues, including those of the catalytically important Walker B and the switch loop motifs, are fundamentally different in the solitary NBDs compared to those in the intact transport protein. The structures reveal crucial interdomain contacts that need to be established for the proper assembly of the functional transporter complex.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Ligação de Hidrogênio , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Transporters of the solute carrier superfamily (SLCs) are responsible for the transmembrane traffic of the majority of chemical substances in cells and tissues and are therefore of fundamental biological importance. As is often the case with membrane proteins that can be heavily glycosylated, a lack of reliable high-affinity binders hinders their functional analysis. Purifying and reconstituting transmembrane proteins in their lipidic environments remains challenging and standard approaches to generate binders for multi-transmembrane proteins, such as SLCs, channels or G protein-coupled receptors (GPCRs) are lacking. While generating protein binders to 27 SLCs, we produced full length protein or cell lines as input material for binder generation by selected binder generation platforms. As a result, we obtained 525 binders for 22 SLCs. We validated the binders with a cell-based validation workflow using immunofluorescent and immunoprecipitation methods to process all obtained binders. Finally, we demonstrated the potential applications of the binders that passed our validation pipeline in structural, biochemical, and biological applications using the exemplary protein SLC12A6, an ion transporter relevant in human disease. With this work, we were able to generate easily renewable and highly specific binders against SLCs, which will greatly facilitate the study of this neglected protein family. We hope that the process will serve as blueprint for the generation of binders against the entire superfamily of SLC transporters.
Assuntos
Ligação Proteica , Proteínas Carreadoras de Solutos , Humanos , Proteínas Carreadoras de Solutos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/química , Células HEK293RESUMO
Fragments of large membrane proteins have the potential to facilitate structural analysis by NMR, but their folding state remains a concern. Here we determined the quality of folding upon heterologous expression for a series of N- or C-terminally truncated fragments of the human Y4 G-protein coupled receptor, amounting to six different complementation pairs. As the individual fragments lack a specific function that could be used to ascertain proper folding, we instead assessed folding on a basic level by studying their membrane topology and by comparing it to well-established structural models of GPCRs. The topology of the fragments was determined using a reporter assay based on C-terminal green fluorescent protein- or alkaline phosphatase-fusions. N-terminal fusions to Lep or Mistic were used if a periplasmic orientation of the N-terminus of the fragments was expected based on predictions. Fragments fused to Mistic expressed at comparably high levels, whereas Lep fusions were produced to a much lower extent. Though none of the fragments exclusively adopted one orientation, often the correct topology predominated. In addition, systematic analysis of the fragment series suggested that the C-terminal half of the Y4 receptor is more important for adopting the correct topology than the N-terminal part. Using the detergent dodecylphosphocholine, selected fragments were solubilized from the membrane and proved sufficiently stable to allow purification. Finally, as a first step toward reconstituting a functional receptor from two fragments, we observed a physical interaction between complementing fragments pairs upon co-expression.
Assuntos
Proteínas de Membrana/química , Fragmentos de Peptídeos/química , Receptores de Neuropeptídeo Y/química , Membrana Celular , Proteínas de Fluorescência Verde , Humanos , Ressonância Magnética Nuclear Biomolecular , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Dobramento de Proteína , Proteínas Recombinantes de Fusão/químicaRESUMO
Substrate-binding protein-dependent secondary transporters are widespread in prokaryotes and are represented most frequently by members of the tripartite ATP-independent periplasmic (TRAP) transporter family. Here, we report the membrane reconstitution of a TRAP transporter, the sialic acid-specific SiaPQM system from Haemophilus influenzae, and elucidate its mechanism of energy coupling. Uptake of sialic acid via membrane-reconstituted SiaQM depends on the presence of the sialic acid-binding protein, SiaP, and is driven by the electrochemical sodium gradient. The interaction between SiaP and SiaQM is specific as transport is not reconstituted using the orthologous sialic acid-binding protein VC1779. Importantly, the binding protein also confers directionality on the transporter, and reversal of sialic acid transport from import to export is only possible in the presence of an excess of unliganded SiaP.
Assuntos
Haemophilus influenzae/química , Proteínas de Membrana Transportadoras/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Simportadores/metabolismo , Transporte Biológico , Metabolismo Energético , Ligação Proteica , Especificidade por Substrato , Proteínas Virais/metabolismoRESUMO
Making your (Dsb) connection: the redox pathway bringing reducing equivalents from bacterial cytoplasm, across the inner membrane, to the three reductive Dsb pathways in the otherwise oxidizing periplasm (see scheme; TR=thioredoxin reductase, Trx=thioredoxin) is reconstituted from purified components. Transfer of reducing equivalents across the membrane is demonstrated and underlying mechanistic details are revealed.
Assuntos
Membrana Celular/metabolismo , Citoplasma/metabolismo , Dissulfetos/química , Escherichia coli/metabolismo , Periplasma/metabolismo , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/metabolismo , Oxirredução , Proteolipídeos/metabolismo , Transdução de SinaisRESUMO
SLC23 family members are transporters of either nucleobases or ascorbate. While the mammalian SLC23 ascorbate transporters are sodium-coupled, the non-mammalian nucleobase transporters have been proposed, but not formally shown, to be proton-coupled symporters. This assignment is exclusively based on in vivo transport assays using protonophores. Here, by establishing the first in vitro transport assay for this protein family, we demonstrate that a representative member of the SLC23 nucleobase transporters operates as a uniporter instead. We explain these conflicting assignments by identifying a critical role of uracil phosphoribosyltransferase, the enzyme converting uracil to UMP, in driving uracil uptake in vivo. Detailed characterization of uracil phosphoribosyltransferase reveals that the sharp reduction of uracil uptake in whole cells in presence of protonophores is caused by acidification-induced enzyme inactivation. The SLC23 family therefore consists of both uniporters and symporters in line with the structurally related SLC4 and SLC26 families that have previously been demonstrated to accommodate both transport modes as well.
Assuntos
Transporte Biológico/fisiologia , Transporte de Íons , Proteínas de Membrana Transportadoras/química , Prótons , Animais , Ácido Ascórbico/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Domínio Catalítico , Escherichia coli , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transporte de Nucleobases/química , Proteínas de Transporte de Nucleobases/metabolismo , Pentosiltransferases/química , Pentosiltransferases/metabolismo , Sódio/metabolismo , Simportadores , Uracila/metabolismoRESUMO
Methods for the cloning of large numbers of open reading frames into expression vectors are of critical importance for challenging structural biology projects. Here we describe a system termed fragment exchange (FX) cloning that facilitates the high-throughput generation of expression constructs. The method is based on a class IIS restriction enzyme and negative selection markers. FX cloning combines attractive features of established recombination- and ligation-independent cloning methods: It allows the straightforward transfer of an open reading frame into a variety of expression vectors and is highly efficient and very economic in its use. In addition, FX cloning avoids the common but undesirable feature of significantly extending target open reading frames with cloning related sequences, as it leaves a minimal seam of only a single extra amino acid to either side of the protein. The method has proven to be very robust and suitable for all common pro- and eukaryotic expression systems. It considerably speeds up the generation of expression constructs compared to traditional methods and thus facilitates a broader expression screening.
Assuntos
Biologia/métodos , Clonagem Molecular/métodos , Sequência de Bases , Sítios de Ligação , DNA/genética , DNA/metabolismo , Clivagem do DNA , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Estudos de Viabilidade , Proteínas de Membrana Transportadoras/genética , Fases de Leitura Aberta/genéticaRESUMO
Overexpression of membrane proteins in Escherichia coli frequently leads to the formation of aggregates or inclusion bodies, which is undesirable for most studies. Ideally, one would like to optimize the expression conditions by monitoring simultaneously and rapidly both the amounts of properly folded and aggregated membrane protein, a requirement not met by any of the currently available methods. Here, we describe a simple gel-based approach with green fluorescent protein as folding indicator to detect well folded and aggregated proteins simultaneously. The method allows for rapid screening and, importantly, pinpointing the most likely bottlenecks in protein production.
Assuntos
Clonagem Molecular/métodos , Proteínas de Membrana/genética , Escherichia coli/genética , Expressão Gênica , Proteínas de Fluorescência Verde , Dobramento de Proteína , Controle de QualidadeRESUMO
Gram-negative bacteria are intrinsically resistant against cytotoxic substances by means of their outer membrane and a network of multidrug efflux systems, acting in synergy. Efflux pumps from various superfamilies with broad substrate preferences sequester and pump drugs across the inner membrane to supply the highly polyspecific and powerful tripartite resistance-nodulation-cell division (RND) efflux pumps with compounds to be extruded across the outer membrane barrier. In Escherichia coli, the tripartite efflux system AcrAB-TolC is the archetype RND multiple drug efflux pump complex. The homotrimeric inner membrane component acriflavine resistance B (AcrB) is the drug specificity and energy transduction center for the drug/proton antiport process. Drugs are bound and expelled via a cycle of mainly three consecutive states in every protomer, constituting a flexible alternating access channel system. This review recapitulates the molecular basis of drug and inhibitor binding, including mechanistic insights into drug efflux by AcrB. It also summarizes 17 years of mutational analysis of the gene acrB, reporting the effect of every substitution on the ability of E. coli to confer resistance toward antibiotics (http://goethe.link/AcrBsubstitutions). We emphasize the functional robustness of AcrB toward single-site substitutions and highlight regions that are more sensitive to perturbation.
Assuntos
Antibacterianos/metabolismo , Farmacorresistência Bacteriana Múltipla/fisiologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Animais , Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Humanos , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
The selective immobilization of proteins represents an essential step in the selection of binding proteins such as antibodies. The immobilization strategy determines how the target protein is presented to the binders and thereby directly affects the experimental outcome. This poses specific challenges for membrane proteins due to their inherent lack of stability and limited exposed hydrophilic surfaces. Here we detail methodologies for the selective immobilization of membrane proteins based on the strong biotin-avidin interaction and with a specific focus on its application for the selection of nanobodies and sybodies. We discuss the challenges in generating and benefits of obtaining an equimolar biotin to target-protein ratio.
Assuntos
Avidina/metabolismo , Biotina/metabolismo , Biotinilação/métodos , Proteínas de Membrana/metabolismo , Anticorpos de Domínio Único/isolamento & purificação , Sequência de Aminoácidos , Avidina/química , Biotina/química , Carbono-Nitrogênio Ligases/química , Carbono-Nitrogênio Ligases/metabolismo , Técnicas de Visualização da Superfície Celular/métodos , Clonagem Molecular/métodos , Escherichia coli , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Klebsiella pneumoniae , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Ligação Proteica , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/metabolismo , Estreptavidina/química , Estreptavidina/metabolismoRESUMO
Here, we provide a protocol to generate synthetic nanobodies, known as sybodies, against any purified protein or protein complex within a 3-week period. Unlike methods that require animals for antibody generation, sybody selections are carried out entirely in vitro under controlled experimental conditions. This is particularly relevant for the generation of conformation-specific binders against labile membrane proteins or protein complexes and allows selections in the presence of non-covalent ligands. Sybodies are especially suited for cases where binder generation via immune libraries fails due to high sequence conservation, toxicity or insufficient stability of the target protein. The procedure entails a single round of ribosome display using the sybody libraries encoded by mRNA, followed by two rounds of phage display and binder identification by ELISA. The protocol is optimized to avoid undesired reduction in binder diversity and enrichment of non-specific binders to ensure the best possible selection outcome. Using the efficient fragment exchange (FX) cloning method, the sybody sequences are transferred from the phagemid to different expression vectors without the need to amplify them by PCR, which avoids unintentional shuffling of complementary determining regions. Using quantitative PCR (qPCR), the efficiency of each selection round is monitored to provide immediate feedback and guide troubleshooting. Our protocol can be carried out by any trained biochemist or molecular biologist using commercially available reagents and typically gives rise to 10-30 unique sybodies exhibiting binding affinities in the range of 500 pM-500 nM.
Assuntos
Técnicas de Química Sintética/métodos , Anticorpos de Domínio Único/química , Bacteriófagos/química , Ribossomos/químicaRESUMO
The SLC26 family of transporters maintains anion equilibria in all kingdoms of life. The family shares a 7 + 7 transmembrane segments inverted repeat architecture with the SLC4 and SLC23 families, but holds a regulatory STAS domain in addition. While the only experimental SLC26 structure is monomeric, SLC26 proteins form structural and functional dimers in the lipid membrane. Here we resolve the structure of an SLC26 dimer embedded in a lipid membrane and characterize its functional relevance by combining PELDOR/DEER distance measurements and biochemical studies with MD simulations and spin-label ensemble refinement. Our structural model reveals a unique interface different from the SLC4 and SLC23 families. The functionally relevant STAS domain is no prerequisite for dimerization. Characterization of heterodimers indicates that protomers in the dimer functionally interact. The combined structural and functional data define the framework for a mechanistic understanding of functional cooperativity in SLC26 dimers.