RESUMO
Mycobacterium tuberculosis is protected from antibiotic therapy by a multi-layered hydrophobic cell envelope. Major facilitator superfamily (MFS) transporter Rv1410 and the periplasmic lipoprotein LprG are involved in transport of triacylglycerides (TAGs) that seal the mycomembrane. Here, we report a 2.7 Å structure of a mycobacterial Rv1410 homologue, which adopts an outward-facing conformation and exhibits unusual transmembrane helix 11 and 12 extensions that protrude ~20 Å into the periplasm. A small, very hydrophobic cavity suitable for lipid transport is constricted by a functionally important ion-lock likely involved in proton coupling. Combining mutational analyses and MD simulations, we propose that TAGs are extracted from the core of the inner membrane into the central cavity via lateral clefts present in the inward-facing conformation. The functional role of the periplasmic helix extensions is to channel the extracted TAG into the lipid binding pocket of LprG.
Assuntos
Proteínas de Membrana Transportadoras , Mycobacterium tuberculosis , Proteínas de Membrana Transportadoras/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Transporte Biológico , Membranas/metabolismo , Lipídeos , Conformação ProteicaRESUMO
Structural studies of G-protein-coupled receptors (GPCRs) are often limited by difficulties in obtaining well-diffracting crystals suitable for high-resolution structure determination. During the past decade, crystallization in lipidic cubic phase (LCP) has become the most successful and widely used technique for obtaining such crystals. Despite often intense efforts, many GPCRs remain refractory to crystallization, even if receptors can be purified in sufficient amounts. To address this issue, we have developed a highly efficient screening and stabilization strategy for GPCRs, based on a fluorescence thermal stability assay readout, which seems to correlate particularly well with those GPCR constructs that remain native during incorporation into the LCP. Detailed protocols are provided for rapid and cost-efficient mutant and construct generation using sequence- and ligation-independent cloning, high-throughput magnetic bead-based protein purification from small-scale expressions in mammalian cells, the screening and optimal combination of mutations for increased receptor thermostability and the rapid identification of suitable chimeric fusion protein constructs for successful crystallization in LCP. We exemplify the method on three receptors from two different classes: the neurokinin 1 receptor, the oxytocin receptor and the parathyroid hormone 1 receptor.
Assuntos
Lipídeos , Receptores Acoplados a Proteínas G , Animais , Cristalização/métodos , Lipídeos/química , Mamíferos , Mutação , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genéticaRESUMO
The neurokinin 1 receptor (NK1R) is involved in inflammation and pain transmission. This pathophysiologically important G proteincoupled receptor is predominantly activated by its cognate agonist substance P (SP) but also by the closely related neurokinins A and B. Here, we report cryoelectron microscopy structures of SP-bound NK1R in complex with its primary downstream signal mediators, Gq and Gs. Our structures reveal how a polar network at the extracellular, solvent-exposed receptor surface shapes the orthosteric pocket and that NK1R adopts a noncanonical active-state conformation with an interface for G protein binding, which is distinct from previously reported structures. Detailed comparisons with antagonist-bound NK1R crystal structures reveal that insurmountable antagonists induce a distinct and long-lasting receptor conformation that sterically blocks SP binding. Together, our structures provide important structural insights into ligand and G protein promiscuity, the lack of basal signaling, and agonist- and antagonist-induced conformations in the neurokinin receptor family.
RESUMO
The peptide hormone oxytocin modulates socioemotional behavior and sexual reproduction via the centrally expressed oxytocin receptor (OTR) across several species. Here, we report the crystal structure of human OTR in complex with retosiban, a nonpeptidic antagonist developed as an oral drug for the prevention of preterm labor. Our structure reveals insights into the detailed interactions between the G protein-coupled receptor (GPCR) and an OTR-selective antagonist. The observation of an extrahelical cholesterol molecule, binding in an unexpected location between helices IV and V, provides a structural rationale for its allosteric effect and critical influence on OTR function. Furthermore, our structure in combination with experimental data allows the identification of a conserved neurohypophyseal receptor-specific coordination site for Mg2+ that acts as potent, positive allosteric modulator for agonist binding. Together, these results further our molecular understanding of the oxytocin/vasopressin receptor family and will facilitate structure-guided development of new therapeutics.
Assuntos
Ocitocina , Receptores de Ocitocina , Sítios de Ligação , Humanos , Ligação Proteica , Receptores de Ocitocina/metabolismoRESUMO
The parathyroid hormone 1 receptor (PTH1R) is a major regulator of mineral ion homeostasis and bone metabolism and is thus considered an attractive drug target for the treatment of disorders in calcium metabolism and bone-related diseases such as osteoporosis. PTH1R is a member of the class B of GPCRs, which all share a dynamic multidomain binding mechanism to the peptide hormone. For a long time, these complexes have been recalcitrant to structural studies despite their great therapeutic relevance. Through extensive engineering of both the receptor and the peptide agonist ligand, we were able to determine the first high-resolution structure of a PTH1R-agonist complex. Comparisons of the PTH1R crystal structure with subsequently reported cryo-electron microscopy structures of the same receptor in complex with a G protein, as well as with other class B GPCR structures bound to antagonists, reveal new insights into the two-step activation mechanism of class B GPCRs and extend our understanding of the precise molecular rearrangements during receptor activation.
Assuntos
Receptor Tipo 1 de Hormônio Paratireóideo/química , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Animais , Microscopia Crioeletrônica , Cristalografia por Raios X , Humanos , Estrutura Secundária de Proteína , Receptor Tipo 1 de Hormônio Paratireóideo/genética , Receptores Acoplados a Proteínas G/genéticaRESUMO
Neurokinins (or tachykinins) are peptides that modulate a wide variety of human physiology through the neurokinin G protein-coupled receptor family, implicated in a diverse array of pathological processes. Here we report high-resolution crystal structures of the human NK1 receptor (NK1R) bound to two small-molecule antagonist therapeutics - aprepitant and netupitant and the progenitor antagonist CP-99,994. The structures reveal the detailed interactions between clinically approved antagonists and NK1R, which induce a distinct receptor conformation resulting in an interhelical hydrogen-bond network that cross-links the extracellular ends of helices V and VI. Furthermore, the high-resolution details of NK1R bound to netupitant establish a structural rationale for the lack of basal activity in NK1R. Taken together, these co-structures provide a comprehensive structural basis of NK1R antagonism and will facilitate the design of new therapeutics targeting the neurokinin receptor family.
Assuntos
Antagonistas dos Receptores de Neurocinina-1/química , Receptores da Neurocinina-1/química , Aprepitanto/química , Aprepitanto/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Antagonistas dos Receptores de Neurocinina-1/farmacologia , Piperidinas/química , Piperidinas/farmacologia , Estrutura Secundária de Proteína , Piridinas/química , Piridinas/farmacologia , Receptores da Neurocinina-1/isolamento & purificação , Receptores da Neurocinina-1/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
Parathyroid hormone 1 receptor (PTH1R) is a class B multidomain G-protein-coupled receptor (GPCR) that controls calcium homeostasis. Two endogenous peptide ligands, parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP), activate the receptor, and their analogs teriparatide and abaloparatide are used in the clinic to increase bone formation as an effective yet costly treatment for osteoporosis. Activation of PTH1R involves binding of the peptide ligand to the receptor extracellular domain (ECD) and transmembrane domain (TMD), a hallmark of class B GPCRs. Here, we present the crystal structure of human PTH1R in complex with a peptide agonist at 2.5-Å resolution, allowing us to delineate the agonist binding mode for this receptor and revealing molecular details within conserved structural motifs that are critical for class B receptor function. Thus, this study provides structural insight into the function of PTH1R and extends our understanding of this therapeutically important class of GPCRs.
Assuntos
Receptor Tipo 1 de Hormônio Paratireóideo/química , Sequência de Aminoácidos , Biomimética , Cristalografia por Raios X , Humanos , Modelos Moleculares , Hormônio Paratireóideo/química , Peptídeos/metabolismo , Ligação ProteicaRESUMO
Despite recent successes, many G protein-coupled receptors (GPCRs) remained refractory to detailed molecular studies due to insufficient production yields, even in the most sophisticated eukaryotic expression systems. Here we introduce a robust method employing directed evolution of GPCRs in yeast that allows fast and efficient generation of receptor variants which show strongly increased functional production levels in eukaryotic expression hosts. Shown by evolving three different receptors in this study, the method is widely applicable, even for GPCRs which are very difficult to express. The evolved variants showed up to a 26-fold increase of functional production in insect cells compared to the wild-type receptors. Next to the increased production, the obtained variants exhibited improved biophysical properties, while functional properties remained largely unaffected. Thus, the presented method broadens the portfolio of GPCRs accessible for detailed investigations. Interestingly, the functional production of GPCRs in yeast can be further increased by induced host adaptation.
Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animais , Western Blotting , Evolução Molecular Direcionada , Humanos , Microscopia de Fluorescência , Receptores Acoplados a Proteínas G/genética , Receptores da Neurocinina-1/genética , Receptores da Neurocinina-1/metabolismo , Receptores de Neurotensina/genética , Receptores de Neurotensina/metabolismo , Receptores Opioides kappa/genética , Receptores Opioides kappa/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/genética , Células Sf9 , SpodopteraRESUMO
Fluorescent probes constitute a valuable toolbox to address a variety of biological questions and they have become irreplaceable for imaging methods. Commonly, such probes consist of fluorescent proteins or small organic fluorophores coupled to biological molecules of interest. Recently, a novel class of fluorescence-based probes, fluorogen-activating proteins (FAPs), has been reported. These binding proteins are based on antibody single-chain variable fragments and activate fluorogenic dyes, which only become fluorescent upon activation and do not fluoresce when free in solution. Here we present a novel class of fluorogen activators, termed FADAs, based on the very robust designed ankyrin repeat protein scaffold, which also readily folds in the reducing environment of the cytoplasm. The FADA generated in this study was obtained by combined selections with ribosome display and yeast surface display. It enhances the fluorescence of malachite green (MG) dyes by a factor of more than 11,000 and thus activates MG to a similar extent as FAPs based on single-chain variable fragments. As shown by structure determination and in vitro measurements, this FADA was evolved to form a homodimer for the activation of MG dyes. Exploiting the favorable properties of the designed ankyrin repeat protein scaffold, we created a FADA biosensor suitable for imaging of proteins on the cell surface, as well as in the cytosol. Moreover, based on the requirement of dimerization for strong fluorogen activation, a prototype FADA biosensor for in situ detection of a target protein and protein-protein interactions was developed. Therefore, FADAs are versatile fluorescent probes that are easily produced and suitable for diverse applications and thus extend the FAP technology.
Assuntos
Repetição de Anquirina , Técnicas Biossensoriais/métodos , Corantes Fluorescentes/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Corantes de Rosanilina/metabolismo , Proteínas Recombinantes/genéticaRESUMO
Agonist binding to G-protein-coupled receptors (GPCRs) triggers signal transduction cascades involving heterotrimeric G proteins as key players. A major obstacle for drug design is the limited knowledge of conformational changes upon agonist binding, the details of interaction with the different G proteins, and the transmission to movements within the G protein. Although a variety of different GPCR/G protein complex structures would be needed, the transient nature of this complex and the intrinsic instability against dissociation make this endeavor very challenging. We have previously evolved GPCR mutants that display higher stability and retain their interaction with G proteins. We aimed at finding all G-protein combinations that preferentially interact with neurotensin receptor 1 (NTR1) and our stabilized mutants. We first systematically analyzed by coimmunoprecipitation the capability of 120 different G-protein combinations consisting of αi1 or αsL and all possible ßγ-dimers to form a heterotrimeric complex. This analysis revealed a surprisingly unrestricted ability of the G-protein subunits to form heterotrimeric complexes, including ßγ-dimers previously thought to be nonexistent, except for combinations containing ß5. A second screen on coupling preference of all G-protein heterotrimers to NTR1 wild type and a stabilized mutant indicated a preference for those Gαi1ßγ combinations containing γ1 and γ11. Heterotrimeric G proteins, including combinations believed to be nonexistent, were purified, and complexes with the GPCR were prepared. Our results shed new light on the combinatorial diversity of G proteins and their coupling to GPCRs and open new approaches to improve the stability of GPCR/G-protein complexes.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Cromatografia em Gel , Proteínas Heterotriméricas de Ligação ao GTP/isolamento & purificação , Humanos , Proteínas Mutantes/metabolismo , Ligação Proteica , Receptores Acoplados a Proteínas G/isolamento & purificação , Células Sf9 , SoluçõesRESUMO
Lipidic cubic phases (LCPs) are used in areas ranging from membrane biology to biodevices. Because some membrane proteins are notoriously unstable at room temperature, and available LCPs undergo transformation to lamellar phases at low temperatures, development of stable low-temperature LCPs for biophysical studies of membrane proteins is called for. Monodihydrosterculin (MDS) is a designer lipid based on monoolein (MO) with a configurationally restricted cyclopropyl ring replacing the olefin. Small-angle X-ray scattering (SAXS) analyses revealed a phase diagram for MDS lacking the high-temperature, highly curved reverse hexagonal phase typical for MO, and extending the cubic phase boundary to lower temperature, thereby establishing the relationship between lipid molecular structure and mesophase behavior. The use of MDS as a new material for LCP-based membrane protein crystallization at low temperature was demonstrated by crystallizing bacteriorhodopsin at 20 °C as well as 4 °C.
Assuntos
Glicerídeos/química , Proteínas de Membrana/química , Bacteriorodopsinas/química , Temperatura Baixa , Cristalização , Proteínas de Membrana/metabolismo , Transição de Fase , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
ATP binding cassette (ABC) transporters mediate vital transport processes in every living cell. ATP hydrolysis, which fuels transport, displays positive cooperativity in numerous ABC transporters. In particular, heterodimeric ABC exporters exhibit pronounced allosteric coupling between a catalytically impaired degenerate site, where nucleotides bind tightly, and a consensus site, at which ATP is hydrolyzed in every transport cycle. Whereas the functional phenomenon of cooperativity is well described, its structural basis remains poorly understood. Here, we present the apo structure of the heterodimeric ABC exporter TM287/288 and compare it to the previously solved structure with adenosine 5'-(ß,γ-imido)triphosphate (AMP-PNP) bound at the degenerate site. In contrast to other ABC exporter structures, the nucleotide binding domains (NBDs) of TM287/288 remain in molecular contact even in the absence of nucleotides, and the arrangement of the transmembrane domains (TMDs) is not influenced by AMP-PNP binding, a notion confirmed by double electron-electron resonance (DEER) measurements. Nucleotide binding at the degenerate site results in structural rearrangements, which are transmitted to the consensus site via two D-loops located at the NBD interface. These loops owe their name from a highly conserved aspartate and are directly connected to the catalytically important Walker B motif. The D-loop at the degenerate site ties the NBDs together even in the absence of nucleotides and substitution of its aspartate by alanine is well-tolerated. By contrast, the D-loop of the consensus site is flexible and the aspartate to alanine mutation and conformational restriction by cross-linking strongly reduces ATP hydrolysis and substrate transport.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Monofosfato de Adenosina/química , Trifosfato de Adenosina/química , Proteínas de Bactérias/química , Lactococcus lactis/química , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/genética , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Regulação Alostérica/fisiologia , Sítio Alostérico , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico Ativo/fisiologia , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
BCL-W is a member of the BCL-2 family of anti-apoptotic proteins. A key event in the regulation of apoptosis is the heterodimerization between anti-apoptotic and pro-apoptotic family members, which involves a conserved surface-exposed groove on the anti-apoptotic proteins. Crystal structures of the ligand binding-competent conformation exist for all anti-apoptotic family members, with the exception of BCL-W, due to the flexibility of the BCL-W groove region. Existing structures had suggested major deviations of the BCL-W groove region from the otherwise structurally highly related remaining anti-apoptotic family members. To capture its ligand binding-competent conformation by counteracting the conformational flexibility of the BCL-W groove, we had selected high-affinity groove-binding designed ankyrin repeat proteins (DARPins) using ribosome display. We now determined two high-resolution crystal structures of human BCL-W in complex with different DARPins at resolutions 1.5 and 1.85Å, in which the structure of BCL-W is virtually identical, and BCL-W adopts a conformation extremely similar to the ligand-free conformation of its closest relative BCL-XL in both structures. However, distinct differences to all previous BCL-W structures are evident, notably in the ligand-binding region. We provide the first structural explanation for the conformational flexibility of the BCL-W groove region in comparison to other BCL-2 family members. Due to the importance of the anti-apoptotic BCL-2 family as drug targets, the presented crystal structure of ligand binding-competent BCL-W may serve as a valuable basis for structure-based drug design in the future and provides a missing piece for the structural characterization of this protein family.
Assuntos
Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ligação Proteica , Conformação ProteicaRESUMO
Antibodies are the most versatile binding proteins in nature with six loops creating a flexible continuous interaction surface. However, in some molecular formats, antibodies are aggregation prone. Designed ankyrin repeat proteins (DARPins) were successfully created as alternative design solutions. Nevertheless, their concave shape, rigidity and incompletely randomized binding surface may limit the epitopes that can be targeted by this extremely stable scaffold. Combining conformational diversity and a continuous convex paratope found in many antibodies with the beneficial biophysical properties of DARPins, we created LoopDARPins, a next generation of DARPins with extended epitope binding properties. We employed X-ray structure determination of a LoopDARPin for design validation. Biophysical characterizations show that the introduction of an elongated loop through consensus design does not decrease the stability of the scaffold,consistent with molecular dynamics simulations. Ribosome-display selections against extracellular signal-regulated kinase 2 (ERK2) and four members of the BCL-2 family (BCL-2, BCL-XL, BCL-W and MCL-1) of anti-apoptotic regulators yielded LoopDARPins with affinities in the mid-picomolar to low nanomol arrange against all targets. The BCL-2 family binders block the interaction with their natural interaction partner and will be valuable reagents to test the apoptotic response in functional assays. With the LoopDARPin scaffold, binders for BCL-2 with an affinity of 30 pM were isolated with only a single round of ribosome display,an enrichment that has not been described for any scaffold. Identical stringent one-round selections with conventional DARPins without loop yielded no binders. The LoopDARPin scaffold may become a highly valuable tool for biotechnological high-throughput applications.