RESUMO
Mitochondrial ADP/ATP carriers transport ADP into the mitochondrial matrix for ATP synthesis, and ATP out to fuel the cell, by cycling between cytoplasmic-open and matrix-open states. The structure of the cytoplasmic-open state is known, but it has proved difficult to understand the transport mechanism in the absence of a structure in the matrix-open state. Here, we describe the structure of the matrix-open state locked by bongkrekic acid bound in the ADP/ATP-binding site at the bottom of the central cavity. The cytoplasmic side of the carrier is closed by conserved hydrophobic residues, and a salt bridge network, braced by tyrosines. Glycine and small amino acid residues allow close-packing of helices on the matrix side. Uniquely, the carrier switches between states by rotation of its three domains about a fulcrum provided by the substrate-binding site. Because these features are highly conserved, this mechanism is likely to apply to the whole mitochondrial carrier family. VIDEO ABSTRACT.
Assuntos
Mitocôndrias/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Translocases Mitocondriais de ADP e ATP/ultraestrutura , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Transporte Biológico , Ácido Bongcréquico/metabolismo , Citoplasma/metabolismo , Mitocôndrias/fisiologia , Translocases Mitocondriais de ADP e ATP/fisiologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Proteínas de Transporte da Membrana Mitocondrial/ultraestrutura , Modelos Moleculares , Conformação Proteica , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The κ-opioid receptor (KOP) mediates the actions of opioids with hallucinogenic, dysphoric, and analgesic activities. The design of KOP analgesics devoid of hallucinatory and dysphoric effects has been hindered by an incomplete structural and mechanistic understanding of KOP agonist actions. Here, we provide a crystal structure of human KOP in complex with the potent epoxymorphinan opioid agonist MP1104 and an active-state-stabilizing nanobody. Comparisons between inactive- and active-state opioid receptor structures reveal substantial conformational changes in the binding pocket and intracellular and extracellular regions. Extensive structural analysis and experimental validation illuminate key residues that propagate larger-scale structural rearrangements and transducer binding that, collectively, elucidate the structural determinants of KOP pharmacology, function, and biased signaling. These molecular insights promise to accelerate the structure-guided design of safer and more effective κ-opioid receptor therapeutics.
Assuntos
Simulação de Acoplamento Molecular , Receptores Opioides kappa/química , Analgésicos/química , Analgésicos/farmacologia , Animais , Sítios de Ligação , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Morfinanos/química , Morfinanos/farmacologia , Ligação Proteica , Estabilidade Proteica , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/metabolismo , Células Sf9 , SpodopteraRESUMO
The Sonic Hedgehog (SHH) morphogen pathway is fundamental for embryonic development and stem cell maintenance and is implicated in various cancers. A key step in signaling is transfer of a palmitate group to the SHH N terminus, catalyzed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT). We present the high-resolution cryo-EM structure of HHAT bound to substrate analog palmityl-coenzyme A and a SHH-mimetic megabody, revealing a heme group bound to HHAT that is essential for HHAT function. A structure of HHAT bound to potent small-molecule inhibitor IMP-1575 revealed conformational changes in the active site that occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the mechanism by which HHAT adapts the membrane environment to transfer an acyl chain across the endoplasmic reticulum membrane. This structure of a membrane-bound O-acyltransferase (MBOAT) superfamily member provides a blueprint for other protein-substrate MBOATs and a template for future drug discovery.
Assuntos
Aciltransferases/antagonistas & inibidores , Aciltransferases/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas Hedgehog/metabolismo , Proteínas de Membrana/metabolismo , Acilação , Aciltransferases/genética , Aciltransferases/ultraestrutura , Regulação Alostérica , Animais , Células COS , Domínio Catalítico , Chlorocebus aethiops , Microscopia Crioeletrônica , Células HEK293 , Heme/metabolismo , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/ultraestrutura , Simulação de Dinâmica Molecular , Palmitoil Coenzima A/metabolismo , Conformação Proteica , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
The liver takes up bile salts from blood to generate bile, enabling absorption of lipophilic nutrients and excretion of metabolites and drugs1. Human Na+-taurocholate co-transporting polypeptide (NTCP) is the main bile salt uptake system in liver. NTCP is also the cellular entry receptor of human hepatitis B and D viruses2,3 (HBV/HDV), and has emerged as an important target for antiviral drugs4. However, the molecular mechanisms underlying NTCP transport and viral receptor functions remain incompletely understood. Here we present cryo-electron microscopy structures of human NTCP in complexes with nanobodies, revealing key conformations of its transport cycle. NTCP undergoes a conformational transition opening a wide transmembrane pore that serves as the transport pathway for bile salts, and exposes key determinant residues for HBV/HDV binding to the outside of the cell. A nanobody that stabilizes pore closure and inward-facing states impairs recognition of the HBV/HDV receptor-binding domain preS1, demonstrating binding selectivity of the viruses for open-to-outside over inward-facing conformations of the NTCP transport cycle. These results provide molecular insights into NTCP 'gated-pore' transport and HBV/HDV receptor recognition mechanisms, and are expected to help with development of liver disease therapies targeting NTCP.
Assuntos
Ácidos e Sais Biliares , Microscopia Crioeletrônica , Fígado , Transportadores de Ânions Orgânicos Dependentes de Sódio , Sódio , Simportadores , Bile/metabolismo , Ácidos e Sais Biliares/metabolismo , Vírus da Hepatite B/metabolismo , Vírus Delta da Hepatite/metabolismo , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/química , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/ultraestrutura , Conformação Proteica , Receptores Virais/metabolismo , Anticorpos de Domínio Único , Sódio/metabolismo , Simportadores/química , Simportadores/metabolismo , Simportadores/ultraestrutura , Internalização do VírusRESUMO
Hyaluronan is an acidic heteropolysaccharide comprising alternating N-acetylglucosamine and glucuronic acid sugars that is ubiquitously expressed in the vertebrate extracellular matrix1. The high-molecular-mass polymer modulates essential physiological processes in health and disease, including cell differentiation, tissue homeostasis and angiogenesis2. Hyaluronan is synthesized by a membrane-embedded processive glycosyltransferase, hyaluronan synthase (HAS), which catalyses the synthesis and membrane translocation of hyaluronan from uridine diphosphate-activated precursors3,4. Here we describe five cryo-electron microscopy structures of a viral HAS homologue at different states during substrate binding and initiation of polymer synthesis. Combined with biochemical analyses and molecular dynamics simulations, our data reveal how HAS selects its substrates, hydrolyses the first substrate to prime the synthesis reaction, opens a hyaluronan-conducting transmembrane channel, ensures alternating substrate polymerization and coordinates hyaluronan inside its transmembrane pore. Our research suggests a detailed model for the formation of an acidic extracellular heteropolysaccharide and provides insights into the biosynthesis of one of the most abundant and essential glycosaminoglycans in the human body.
Assuntos
Hialuronan Sintases , Ácido Hialurônico , Phycodnaviridae , Microscopia Crioeletrônica , Hialuronan Sintases/metabolismo , Phycodnaviridae/enzimologia , PolímerosRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Despite much effort, antibody therapies for Alzheimer's disease (AD) have shown limited efficacy. Challenges to the rational design of effective antibodies include the difficulty of achieving specific affinity to critical targets, poor expression, and antibody aggregation caused by buried charges and unstructured loops. To overcome these challenges, we grafted previously determined sequences of fibril-capping amyloid inhibitors onto a camel heavy chain antibody scaffold. These sequences were designed to cap fibrils of tau, known to form the neurofibrillary tangles of AD, thereby preventing fibril elongation. The nanobodies grafted with capping inhibitors blocked tau aggregation in biosensor cells seeded with postmortem brain extracts from AD and progressive supranuclear palsy (PSP) patients. The tau capping nanobody inhibitors also blocked seeding by recombinant tau oligomers. Another challenge to the design of effective antibodies is their poor blood-brain barrier (BBB) penetration. In this study, we also designed a bispecific nanobody composed of a nanobody that targets a receptor on the BBB and a tau capping nanobody inhibitor, conjoined by a flexible linker. We provide evidence that the bispecific nanobody improved BBB penetration over the tau capping inhibitor alone after intravenous administration in mice. Our results suggest that the design of synthetic antibodies that target sequences that drive protein aggregation may be a promising approach to inhibit the prion-like seeding of tau and other proteins involved in AD and related proteinopathies.
Assuntos
Doença de Alzheimer , Anticorpos de Domínio Único , Paralisia Supranuclear Progressiva , Humanos , Animais , Camundongos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Proteínas tau/metabolismo , Anticorpos de Domínio Único/farmacologia , Anticorpos de Domínio Único/metabolismo , Emaranhados Neurofibrilares/metabolismo , Paralisia Supranuclear Progressiva/metabolismo , Anticorpos/metabolismo , Encéfalo/metabolismoRESUMO
Type A γ-aminobutyric acid (GABAA) receptors are pentameric ligand-gated ion channels and the main drivers of fast inhibitory neurotransmission in the vertebrate nervous system1,2. Their dysfunction is implicated in a range of neurological disorders, including depression, epilepsy and schizophrenia3,4. Among the numerous assemblies that are theoretically possible, the most prevalent in the brain are the α1ß2/3γ2 GABAA receptors5. The ß3 subunit has an important role in maintaining inhibitory tone, and the expression of this subunit alone is sufficient to rescue inhibitory synaptic transmission in ß1-ß3 triple knockout neurons6. So far, efforts to generate accurate structural models for heteromeric GABAA receptors have been hampered by the use of engineered receptors and the presence of detergents7-9. Notably, some recent cryo-electron microscopy reconstructions have reported 'collapsed' conformations8,9; however, these disagree with the structure of the prototypical pentameric ligand-gated ion channel the Torpedo nicotinic acetylcholine receptor10,11, the large body of structural work on homologous homopentameric receptor variants12 and the logic of an ion-channel architecture. Here we present a high-resolution cryo-electron microscopy structure of the full-length human α1ß3γ2L-a major synaptic GABAA receptor isoform-that is functionally reconstituted in lipid nanodiscs. The receptor is bound to a positive allosteric modulator 'megabody' and is in a desensitized conformation. Each GABAA receptor pentamer contains two phosphatidylinositol-4,5-bisphosphate molecules, the head groups of which occupy positively charged pockets in the intracellular juxtamembrane regions of α1 subunits. Beyond this level, the intracellular M3-M4 loops are largely disordered, possibly because interacting post-synaptic proteins are not present. This structure illustrates the molecular principles of heteromeric GABAA receptor organization and provides a reference framework for future mechanistic investigations of GABAergic signalling and pharmacology.
Assuntos
Microscopia Crioeletrônica , Bicamadas Lipídicas/química , Receptores de GABA-A/química , Receptores de GABA-A/ultraestrutura , Regulação Alostérica , Sequência de Aminoácidos , Sítios de Ligação , Condutividade Elétrica , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/ultraestrutura , Estrutura Quaternária de Proteína , Receptores de GABA-A/metabolismoRESUMO
In Fig. 5b, d, the arrows showing transmembrane domain rotations were inadvertently pointing clockwise instead of anticlockwise. Similarly, 'anticlockwise' should have been 'clockwise' in the sentence 'This conformational change of the ECD triggers a clockwise rotation of the TMD.' In Extended Data Table 1, the units of the column 'Model resolution' should have been Å instead of Å2. These errors have been corrected online.
RESUMO
Type-A γ-aminobutyric (GABAA) receptors are ligand-gated chloride channels with a very rich pharmacology. Some of their modulators, including benzodiazepines and general anaesthetics, are among the most successful drugs in clinical use and are common substances of abuse. Without reliable structural data, the mechanistic basis for the pharmacological modulation of GABAA receptors remains largely unknown. Here we report several high-resolution cryo-electron microscopy structures in which the full-length human α1ß3γ2L GABAA receptor in lipid nanodiscs is bound to the channel-blocker picrotoxin, the competitive antagonist bicuculline, the agonist GABA (γ-aminobutyric acid), and the classical benzodiazepines alprazolam and diazepam. We describe the binding modes and mechanistic effects of these ligands, the closed and desensitized states of the GABAA receptor gating cycle, and the basis for allosteric coupling between the extracellular, agonist-binding region and the transmembrane, pore-forming region. This work provides a structural framework in which to integrate previous physiology and pharmacology research and a rational basis for the development of GABAA receptor modulators.
Assuntos
Alprazolam/química , Bicuculina/química , Microscopia Crioeletrônica , Diazepam/química , Picrotoxina/química , Receptores de GABA-A/química , Receptores de GABA-A/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação Alostérica/efeitos dos fármacos , Alprazolam/farmacologia , Benzodiazepinas/química , Benzodiazepinas/farmacologia , Bicuculina/farmacologia , Ligação Competitiva/efeitos dos fármacos , Diazepam/farmacologia , Moduladores GABAérgicos/química , Moduladores GABAérgicos/farmacologia , Humanos , Ligantes , Modelos Moleculares , Nanoestruturas/química , Picrotoxina/farmacologiaRESUMO
Influenza A viruses are responsible for seasonal epidemics, and pandemics can arise from the transmission of novel zoonotic influenza A viruses to humans1,2. Influenza A viruses contain a segmented negative-sense RNA genome, which is transcribed and replicated by the viral-RNA-dependent RNA polymerase (FluPolA) composed of PB1, PB2 and PA subunits3-5. Although the high-resolution crystal structure of FluPolA of bat influenza A virus has previously been reported6, there are no complete structures available for human and avian FluPolA. Furthermore, the molecular mechanisms of genomic viral RNA (vRNA) replication-which proceeds through a complementary RNA (cRNA) replicative intermediate, and requires oligomerization of the polymerase7-10-remain largely unknown. Here, using crystallography and cryo-electron microscopy, we determine the structures of FluPolA from human influenza A/NT/60/1968 (H3N2) and avian influenza A/duck/Fujian/01/2002 (H5N1) viruses at a resolution of 3.0-4.3 Å, in the presence or absence of a cRNA or vRNA template. In solution, FluPolA forms dimers of heterotrimers through the C-terminal domain of the PA subunit, the thumb subdomain of PB1 and the N1 subdomain of PB2. The cryo-electron microscopy structure of monomeric FluPolA bound to the cRNA template reveals a binding site for the 3' cRNA at the dimer interface. We use a combination of cell-based and in vitro assays to show that the interface of the FluPolA dimer is required for vRNA synthesis during replication of the viral genome. We also show that a nanobody (a single-domain antibody) that interferes with FluPolA dimerization inhibits the synthesis of vRNA and, consequently, inhibits virus replication in infected cells. Our study provides high-resolution structures of medically relevant FluPolA, as well as insights into the replication mechanisms of the viral RNA genome. In addition, our work identifies sites in FluPolA that could be targeted in the development of antiviral drugs.
Assuntos
Genoma Viral/genética , Vírus da Influenza A Subtipo H3N2/enzimologia , Virus da Influenza A Subtipo H5N1/enzimologia , Modelos Moleculares , RNA Polimerase Dependente de RNA/química , Microscopia Crioeletrônica , Cristalização , Estrutura Terciária de Proteína , Anticorpos de Domínio Único/metabolismo , Replicação ViralRESUMO
The surname of author Toon Laeremans was misspelled 'Laermans'. This error has been corrected online.
RESUMO
Metabotropic glutamate receptors are family C G-protein-coupled receptors. They form obligate dimers and possess extracellular ligand-binding Venus flytrap domains, which are linked by cysteine-rich domains to their 7-transmembrane domains. Spectroscopic studies show that signalling is a dynamic process, in which large-scale conformational changes underlie the transmission of signals from the extracellular Venus flytraps to the G protein-coupling domains-the 7-transmembrane domains-in the membrane. Here, using a combination of X-ray crystallography, cryo-electron microscopy and signalling studies, we present a structural framework for the activation mechanism of metabotropic glutamate receptor subtype 5. Our results show that agonist binding at the Venus flytraps leads to a compaction of the intersubunit dimer interface, thereby bringing the cysteine-rich domains into close proximity. Interactions between the cysteine-rich domains and the second extracellular loops of the receptor enable the rigid-body repositioning of the 7-transmembrane domains, which come into contact with each other to initiate signalling.
Assuntos
Receptor de Glutamato Metabotrópico 5/química , Receptor de Glutamato Metabotrópico 5/metabolismo , Transdução de Sinais , Regulação Alostérica , Microscopia Crioeletrônica , Cristalografia por Raios X , Cisteína/química , Cisteína/metabolismo , Humanos , Ligantes , Modelos Moleculares , Domínios Proteicos , Estabilidade Proteica , Receptor de Glutamato Metabotrópico 5/ultraestruturaRESUMO
Mutations in the gene coding for leucine-rich repeat kinase 2 (LRRK2) are a leading cause of the inherited form of Parkinson's disease (PD), while LRRK2 overactivation is also associated with the more common idiopathic form of PD. LRRK2 is a large multidomain protein, including a GTPase as well as a Ser/Thr protein kinase domain. Common, disease-causing mutations increase LRRK2 kinase activity, presenting LRRK2 as an attractive target for drug discovery. Currently, drug development has mainly focused on ATP-competitive kinase inhibitors. Here, we report the identification and characterization of a variety of nanobodies that bind to different LRRK2 domains and inhibit or activate LRRK2 in cells and in in vitro. Importantly, nanobodies were identified that inhibit LRRK2 kinase activity while binding to a site that is topographically distinct from the active site and thus act through an allosteric inhibitory mechanism that does not involve binding to the ATP pocket or even to the kinase domain. Moreover, while certain nanobodies completely inhibit the LRRK2 kinase activity, we also identified nanobodies that specifically inhibit the phosphorylation of Rab protein substrates. Finally, in contrast to current type I kinase inhibitors, the studied kinase-inhibitory nanobodies did not induce LRRK2 microtubule association. These comprehensively characterized nanobodies represent versatile tools to study the LRRK2 function and mechanism and can pave the way toward novel diagnostic and therapeutic strategies for PD.
Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Doença de Parkinson/metabolismo , Anticorpos de Domínio Único , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Animais , Sítios de Ligação , Mapeamento de Epitopos , Células HEK293 , Humanos , Camundongos , Microtúbulos/metabolismo , Fosforilação , Ligação Proteica , Células RAW 264.7 , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Salmonella enterica serovar Typhimurium melibiose permease (MelBSt) is a prototype of the Na+-coupled major facilitator superfamily transporters, which are important for the cellular uptake of molecules including sugars and small drugs. Although the symport mechanisms have been well-studied, mechanisms of substrate binding and translocation remain enigmatic. We have previously determined the sugar-binding site of outward-facing MelBSt by crystallography. To obtain other key kinetic states, here we raised camelid single-domain nanobodies (Nbs) and carried out a screening against the WT MelBSt under 4 ligand conditions. We applied an in vivo cAMP-dependent two-hybrid assay to detect interactions of Nbs with MelBSt and melibiose transport assays to determine the effects on MelBSt functions. We found that all selected Nbs showed partial to complete inhibitions of MelBSt transport activities, confirming their intracellular interactions. A group of Nbs (714, 725, and 733) was purified, and isothermal titration calorimetry measurements showed that their binding affinities were significantly inhibited by the substrate melibiose. When titrating melibiose to the MelBSt/Nb complexes, Nb also inhibited the sugar-binding. However, the Nb733/MelBSt complex retained binding to the coupling cation Na+ and also to the regulatory enzyme EIIAGlc of the glucose-specific phosphoenolpyruvate/sugar phosphotransferase system. Further, EIIAGlc/MelBSt complex also retained binding to Nb733 and formed a stable supercomplex. All data indicated that MelBSt trapped by Nbs retained its physiological functions and the trapped conformation is similar to that bound by the physiological regulator EIIAGlc. Therefore, these conformational Nbs can be useful tools for further structural, functional, and conformational analyses.
Assuntos
Anticorpos de Domínio Único , Simportadores , Anticorpos de Domínio Único/metabolismo , Melibiose/metabolismo , Simportadores/metabolismo , Transporte de Íons , Sódio/metabolismoRESUMO
Nanobodies are popular and versatile tools for structural biology. They have a compact single immunoglobulin domain organization, bind target proteins with high affinities while reducing their conformational heterogeneity and stabilize multi-protein complexes. Here we demonstrate that engineered nanobodies can also help overcome two major obstacles that limit the resolution of single-particle cryo-electron microscopy reconstructions: particle size and preferential orientation at the water-air interfaces. We have developed and characterized constructs, termed megabodies, by grafting nanobodies onto selected protein scaffolds to increase their molecular weight while retaining the full antigen-binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we demonstrate that megabodies can be used to obtain three-dimensional reconstructions for membrane proteins that suffer from severe preferential orientation or are otherwise too small to allow accurate particle alignment.
Assuntos
Microscopia Crioeletrônica/métodos , Lipídeos/química , Complexos Multiproteicos/química , Receptores de GABA-A/química , Imagem Individual de Molécula/métodos , Análise de Célula Única/métodos , Anticorpos de Domínio Único/química , Humanos , Modelos Moleculares , Estrutura Molecular , Conformação ProteicaRESUMO
Canonical fibroblast growth factors (FGFs) activate FGF receptors (FGFRs) through paracrine or autocrine mechanisms in a process that requires cooperation with heparan sulfate proteoglycans, which function as co-receptors for FGFR activation. By contrast, endocrine FGFs (FGF19, FGF21 and FGF23) are circulating hormones that regulate critical metabolic processes in a variety of tissues. FGF19 regulates bile acid synthesis and lipogenesis, whereas FGF21 stimulates insulin sensitivity, energy expenditure and weight loss. Endocrine FGFs signal through FGFRs in a manner that requires klothos, which are cell-surface proteins that possess tandem glycosidase domains. Here we describe the crystal structures of free and ligand-bound ß-klotho extracellular regions that reveal the molecular mechanism that underlies the specificity of FGF21 towards ß-klotho and demonstrate how the FGFR is activated in a klotho-dependent manner. ß-Klotho serves as a primary 'zip code'-like receptor that acts as a targeting signal for FGF21, and FGFR functions as a catalytic subunit that mediates intracellular signalling. Our structures also show how the sugar-cutting enzyme glycosidase has evolved to become a specific receptor for hormones that regulate metabolic processes, including the lowering of blood sugar levels. Finally, we describe an agonistic variant of FGF21 with enhanced biological activity and present structural insights into the potential development of therapeutic agents for diseases linked to endocrine FGFs.
Assuntos
Fatores de Crescimento de Fibroblastos/química , Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Transdução de Sinais , Sítios de Ligação , Cristalografia por Raios X , Espaço Extracelular/metabolismo , Fator de Crescimento de Fibroblastos 23 , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Células HEK293 , Humanos , Proteínas Klotho , Ligantes , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Especificidade por SubstratoRESUMO
The protein kinase Akt is one of the primary effectors of growth factor signaling in the cell. Akt responds specifically to the lipid second messengers phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] via its PH domain, leading to phosphorylation of its activation loop and the hydrophobic motif of its kinase domain, which are critical for activity. We have now determined the crystal structure of Akt1, revealing an autoinhibitory interface between the PH and kinase domains that is often mutated in cancer and overgrowth disorders. This interface persists even after stoichiometric phosphorylation, thereby restricting maximum Akt activity to PI(3,4,5)P3- or PI(3,4)P2-containing membranes. Our work helps to resolve the roles of lipids and phosphorylation in the activation of Akt and has wide implications for the spatiotemporal control of Akt and potentially lipid-activated kinase signaling in general.
Assuntos
Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , Animais , Sítios de Ligação , Humanos , Insetos , Metabolismo dos Lipídeos , Fosfatos de Fosfatidilinositol/genética , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Proteínas Proto-Oncogênicas c-akt/genética , Piruvato Desidrogenase Quinase de Transferência de Acetil/genética , Células Sf9RESUMO
With conformation-specific nanobodies being used for a wide range of structural, biochemical, and cell biological applications, there is a demand for antigen-binding fragments (Fabs) that specifically and tightly bind these nanobodies without disturbing the nanobody-target protein interaction. Here, we describe the development of a synthetic Fab (termed NabFab) that binds the scaffold of an alpaca-derived nanobody with picomolar affinity. We demonstrate that upon complementary-determining region grafting onto this parent nanobody scaffold, nanobodies recognizing diverse target proteins and derived from llama or camel can cross-react with NabFab without loss of affinity. Using NabFab as a fiducial and size enhancer (50 kDa), we determined the high-resolution cryogenic electron microscopy (cryo-EM) structures of nanobody-bound VcNorM and ScaDMT, both small membrane proteins of â¼50 kDa. Using an additional anti-Fab nanobody further facilitated reliable initial three-dimensional structure determination from small cryo-EM test datasets. Given that NabFab is of synthetic origin, is humanized, and can be conveniently expressed in Escherichia coli in large amounts, it may be useful not only for structural biology but also for biomedical applications.
Assuntos
Microscopia Crioeletrônica/métodos , Fragmentos Fab das Imunoglobulinas/química , Proteínas de Membrana/química , Anticorpos de Domínio Único/química , Animais , Camelídeos Americanos , Camelus , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Microscopia Eletrônica , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Alinhamento de Sequência , Análise de Sequência de ProteínaRESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is essential to maintain fluid homeostasis in key organs. Functional impairment of CFTR due to mutations in the cftr gene leads to cystic fibrosis. Here, we show that the first nucleotide-binding domain (NBD1) of CFTR can spontaneously adopt an alternate conformation that departs from the canonical NBD fold previously observed. Crystallography reveals that this conformation involves a topological reorganization of NBD1. Single-molecule fluorescence resonance energy transfer microscopy shows that the equilibrium between the conformations is regulated by adenosine triphosphate binding. However, under destabilizing conditions, such as the disease-causing mutation F508del, this conformational flexibility enables unfolding of the ß-subdomain. Our data indicate that, in wild-type CFTR, this conformational transition of NBD1 regulates channel function, but, in the presence of the F508del mutation, it allows domain misfolding and subsequent protein degradation. Our work provides a framework to design conformation-specific therapeutics to prevent noxious transitions.