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1.
Proc Natl Acad Sci U S A ; 118(47)2021 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-34782475

RESUMO

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 ∼50 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.

2.
Acta Crystallogr F Struct Biol Commun ; 77(Pt 10): 374-384, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34605442

RESUMO

paaR2-paaA2-parE2 is a three-component toxin-antitoxin module found in prophage CP-993P of Escherichia coli O157:H7. Transcription regulation of this module occurs via the 123-amino-acid regulator PaaR2, which forms a large oligomeric structure. Despite appearing to be well folded, PaaR2 withstands crystallization, as does its N-terminal DNA-binding domain. Native mass spectrometry was used to screen for nanobodies that form a unique complex and stabilize the octameric structure of PaaR2. One such nanobody, Nb33, allowed crystallization of the protein. The resulting crystals belong to space group F432, with unit-cell parameter a = 317 Å, diffract to 4.0 Šresolution and are likely to contain four PaaR2 monomers and four nanobody monomers in the asymmetric unit. Crystals of two truncates containing the N-terminal helix-turn-helix domain also interact with Nb33, and the corresponding co-crystals diffracted to 1.6 and 1.75 Šresolution.

3.
Nat Chem Biol ; 17(9): 989-997, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34341587

RESUMO

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.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/isolamento & purificação , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Modelos Moleculares , Conformação Proteica , Desdobramento de Proteína
4.
Sci Adv ; 7(35)2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34452907

RESUMO

The class IB phosphoinositide 3-kinase (PI3K), PI3Kγ, is a master regulator of immune cell function and a promising drug target for both cancer and inflammatory diseases. Critical to PI3Kγ function is the association of the p110γ catalytic subunit to either a p101 or p84 regulatory subunit, which mediates activation by G protein-coupled receptors. Here, we report the cryo-electron microscopy structure of a heterodimeric PI3Kγ complex, p110γ-p101. This structure reveals a unique assembly of catalytic and regulatory subunits that is distinct from other class I PI3K complexes. p101 mediates activation through its Gßγ-binding domain, recruiting the heterodimer to the membrane and allowing for engagement of a secondary Gßγ-binding site in p110γ. Mutations at the p110γ-p101 and p110γ-adaptor binding domain interfaces enhanced Gßγ activation. A nanobody that specifically binds to the p101-Gßγ interface blocks activation, providing a novel tool to study and target p110γ-p101-specific signaling events in vivo.

5.
Proc Natl Acad Sci U S A ; 118(33)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34385319

RESUMO

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.

6.
Structure ; 2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34348129

RESUMO

There is considerable interest in developing antibodies as modulators of signaling pathways. One of the most important signaling pathways in higher eukaryotes is the phosphoinositide 3-kinase (PI3K) pathway, which plays fundamental roles in growth, metabolism, and immunity. The class IB PI3K, PI3Kγ, is a heterodimeric complex composed of a catalytic p110γ subunit bound to a p101 or p84 regulatory subunit. PI3Kγ is a critical component in multiple immune signaling processes and is dependent on activation by Ras and G protein-coupled receptors (GPCRs) to mediate its cellular roles. Here we describe the rapid and efficient characterization of multiple PI3Kγ binding single-chain camelid nanobodies using hydrogen-deuterium exchange (HDX) mass spectrometry (MS) for structural and biochemical studies. We identify nanobodies that stimulated lipid kinase activity, block Ras activation, and specifically inhibited p101-mediated GPCR activation. Overall, our work reveals insight into PI3Kγ regulation and identifies sites that may be exploited for therapeutic development.

7.
J Cell Biol ; 220(8)2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34132745

RESUMO

Photoreceptors rely on distinct membrane compartments to support their specialized function. Unlike protein localization, identification of critical differences in membrane content has not yet been expanded to lipids, due to the difficulty of isolating domain-specific samples. We have overcome this by using SMA to coimmunopurify membrane proteins and their native lipids from two regions of photoreceptor ROS disks. Each sample's copurified lipids were subjected to untargeted lipidomic and fatty acid analysis. Extensive differences between center (rhodopsin) and rim (ABCA4 and PRPH2/ROM1) samples included a lower PC to PE ratio and increased LC- and VLC-PUFAs in the center relative to the rim region, which was enriched in shorter, saturated FAs. The comparatively few differences between the two rim samples likely reflect specific protein-lipid interactions. High-resolution profiling of the ROS disk lipid composition gives new insights into how intricate membrane structure and protein activity are balanced within the ROS, and provides a model for future studies of other complex cellular structures.


Assuntos
Membrana Celular/metabolismo , Proteínas do Olho/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Animais , Bovinos , Membrana Celular/ultraestrutura , Proteínas do Olho/imunologia , Lipidômica , Proteínas de Membrana/imunologia , Camundongos Endogâmicos BALB C , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Nanotecnologia , Periferinas/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/ultraestrutura , Rodopsina/metabolismo , Anticorpos de Domínio Único/imunologia , Tetraspaninas/metabolismo
8.
Nat Methods ; 18(1): 60-68, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33408403

RESUMO

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 Proteica
9.
Elife ; 92020 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-33349335

RESUMO

Synaptojanin1 (Synj1) is a phosphoinositide phosphatase, important in clathrin uncoating during endocytosis of presynaptic vesicles. It was identified as a potential drug target for Alzheimer's disease, Down syndrome, and TBC1D24-associated epilepsy, while also loss-of-function mutations in Synj1 are associated with epilepsy and Parkinson's disease. Despite its involvement in a range of disorders, structural, and detailed mechanistic information regarding the enzyme is lacking. Here, we report the crystal structure of the 5-phosphatase domain of Synj1. Moreover, we also present a structure of this domain bound to the substrate diC8-PI(3,4,5)P3, providing the first image of a 5-phosphatase with a trapped substrate in its active site. Together with an analysis of the contribution of the different inositide phosphate groups to catalysis, these structures provide new insights in the Synj1 mechanism. Finally, we analysed the effect of three clinical missense mutations (Y793C, R800C, Y849C) on catalysis, unveiling the molecular mechanisms underlying Synj1-associated disease.


Assuntos
Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Humanos , Mutação de Sentido Incorreto , Conformação Proteica , Domínios Proteicos
10.
Elife ; 92020 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-33350937

RESUMO

Energy-coupling factor (ECF) transporters mediate import of micronutrients in prokaryotes. They consist of an integral membrane S-component (that binds substrate) and ECF module (that powers transport by ATP hydrolysis). It has been proposed that different S-components compete for docking onto the same ECF module, but a minimal liposome-reconstituted system, required to substantiate this idea, is lacking. Here, we co-reconstituted ECF transporters for folate (ECF-FolT2) and pantothenate (ECF-PanT) into proteoliposomes, and assayed for crosstalk during active transport. The kinetics of transport showed that exchange of S-components is part of the transport mechanism. Competition experiments suggest much slower substrate association with FolT2 than with PanT. Comparison of a crystal structure of ECF-PanT with previously determined structures of ECF-FolT2 revealed larger conformational changes upon binding of folate than pantothenate, which could explain the kinetic differences. Our work shows that a minimal in vitro system with two reconstituted transporters recapitulates intricate kinetics behaviour observed in vivo.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transporte Biológico Ativo/fisiologia , Técnicas In Vitro , Lactobacillus delbrueckii , Modelos Moleculares , Ligação Proteica , Conformação Proteica
11.
Biophys J ; 119(11): 2205-2218, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33137306

RESUMO

VPS34 complex II (VPS34CII) is a 386-kDa assembly of the lipid kinase subunit VPS34 and three regulatory subunits that altogether function as a prototypical class III phosphatidylinositol-3-kinase (PI3K). When the active VPS34CII complex is docked to the cytoplasmic surface of endosomal membranes, it phosphorylates its substrate lipid (phosphatidylinositol, PI) to generate the essential signaling lipid phosphatidylinositol-3-phosphate (PI3P). In turn, PI3P recruits an array of signaling proteins containing PI3P-specific targeting domains (including FYVE, PX, and PROPPINS) to the membrane surface, where they initiate key cell processes. In endocytosis and early endosome development, net VPS34CII-catalyzed PI3P production is greatly amplified by Rab5A, a small G protein of the Ras GTPase superfamily. Moreover, VPS34CII and Rab5A are each strongly linked to multiple human diseases. Thus, a molecular understanding of the mechanism by which Rab5A activates lipid kinase activity will have broad impacts in both signaling biology and medicine. Two general mechanistic models have been proposed for small G protein activation of PI3K lipid kinases. 1) In the membrane recruitment mechanism, G protein association increases the density of active kinase on the membrane. And 2) in the allosteric activation mechanism, G protein allosterically triggers an increase in the specific activity (turnover rate) of the membrane-bound kinase molecule. This study employs an in vitro single-molecule approach to elucidate the mechanism of GTP-Rab5A-associated VPS34CII kinase activation in a reconstituted GTP-Rab5A-VPS34CII-PI3P-PX signaling pathway on a target membrane surface. The findings reveal that both membrane recruitment and allosteric mechanisms make important contributions to the large increase in VPS34CII kinase activity and PI3P production triggered by membrane-anchored GTP-Rab5A. Notably, under near-physiological conditions in the absence of other activators, membrane-anchored GTP-Rab5A provides strong, virtually binary on-off switching and is required for VPS34CII membrane binding and PI3P production.


Assuntos
Classe III de Fosfatidilinositol 3-Quinases , Endossomos , Proteínas rab5 de Ligação ao GTP , Endocitose , Humanos , Membranas Intracelulares , Fosfatidilinositóis
12.
Proc Natl Acad Sci U S A ; 117(48): 30476-30487, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33214152

RESUMO

None of the current superresolution microscopy techniques can reliably image the changes in endogenous protein nanoclustering dynamics associated with specific conformations in live cells. Single-domain nanobodies have been invaluable tools to isolate defined conformational states of proteins, and we reasoned that expressing these nanobodies coupled to single-molecule imaging-amenable tags could allow superresolution analysis of endogenous proteins in discrete conformational states. Here, we used anti-GFP nanobodies tagged with photoconvertible mEos expressed as intrabodies, as a proof-of-concept to perform single-particle tracking on a range of GFP proteins expressed in live cells, neurons, and small organisms. We next expressed highly specialized nanobodies that target conformation-specific endogenous ß2-adrenoreceptor (ß2-AR) in neurosecretory cells, unveiling real-time mobility behaviors of activated and inactivated endogenous conformers during agonist treatment in living cells. We showed that activated ß2-AR (Nb80) is highly immobile and organized in nanoclusters. The Gαs-GPCR complex detected with Nb37 displayed higher mobility with surprisingly similar nanoclustering dynamics to that of Nb80. Activated conformers are highly sensitive to dynamin inhibition, suggesting selective targeting for endocytosis. Inactivated ß2-AR (Nb60) molecules are also largely immobile but relatively less sensitive to endocytic blockade. Expression of single-domain nanobodies therefore provides a unique opportunity to capture highly transient changes in the dynamic nanoscale organization of endogenous proteins.


Assuntos
Modelos Moleculares , Conformação Proteica , Receptores Adrenérgicos beta 2/química , Imagem Individual de Molécula , Anticorpos de Domínio Único/química , Animais , Linhagem Celular , Endocitose , Imunofluorescência , Expressão Gênica , Genes Reporter , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Camundongos , Ligação Proteica , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Proteínas Recombinantes de Fusão , Imagem Individual de Molécula/métodos , Anticorpos de Domínio Único/metabolismo , Peixe-Zebra
13.
PLoS One ; 15(5): e0232846, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32380514

RESUMO

The structure of lactose permease, stabilized in a periplasmic open conformation by two Gly to Trp replacements (LacYww) and complexed with a nanobody directed against this conformation, provides the highest resolution structure of the symporter. The nanobody binds in a different manner than two other nanobodies made against the same mutant, which also bind to the same general region on the periplasmic side. This region of the protein may represent an immune hotspot. The CDR3 loop of the nanobody is held by hydrogen bonds in a conformation that partially blocks access to the substrate-binding site. As a result, kon and koff for galactoside binding to either LacY or the double mutant complexed with the nanobody are lower than for the other two LacY/nanobody complexes though the Kd values are similar, reflecting the fact that the nanobodies rigidify structures along the pathway. While the wild-type LacY/nanobody complex clearly stabilizes a similar 'extracellular open' conformation in solution, judged by binding kinetics, the complex with wild-type LacY did not yet crystallize, suggesting the nanobody does not bind strongly enough to shift the equilibrium to stabilize a periplasmic side-open conformation suitable for crystallization. However, the similarity of the galactoside binding kinetics for the nanobody-bound complexes with wild type LacY and with LacYWW indicates that they have similar structures, showing that the reported co-structures reliably show nanobody interactions with LacY.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Transporte de Monossacarídeos/química , Anticorpos de Domínio Único/química , Simportadores/química , Substituição de Aminoácidos , Reações Antígeno-Anticorpo , Sítios de Ligação , Cristalografia por Raios X , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/imunologia , Galactose/metabolismo , Glicina/química , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/imunologia , Mutação de Sentido Incorreto , Mutação Puntual , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Anticorpos de Domínio Único/imunologia , Relação Estrutura-Atividade , Simportadores/genética , Simportadores/imunologia , Tiogalactosídeos/química , Triptofano/química
14.
Nat Commun ; 11(1): 1145, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32123179

RESUMO

Recent studies show that GPCRs rapidly interconvert between multiple states although our ability to interrogate, monitor and visualize them is limited by a relative lack of suitable tools. We previously reported two nanobodies (Nb39 and Nb6) that stabilize distinct ligand- and efficacy-delimited conformations of the kappa opioid receptor. Here, we demonstrate via X-ray crystallography a nanobody-targeted allosteric binding site by which Nb6 stabilizes a ligand-dependent inactive state. As Nb39 stabilizes an active-like state, we show how these two state-dependent nanobodies can provide real-time reporting of ligand stabilized states in cells in situ. Significantly, we demonstrate that chimeric GPCRs can be created with engineered nanobody binding sites to report ligand-stabilized states. Our results provide both insights regarding potential mechanisms for allosterically modulating KOR with nanobodies and a tool for reporting the real-time, in situ dynamic range of GPCR activity.


Assuntos
Receptores Opioides kappa/química , Receptores Opioides kappa/metabolismo , Anticorpos de Domínio Único/química , Sítio Alostérico , Sítios de Ligação , Técnicas Biossensoriais , Cristalografia por Raios X , AMP Cíclico/metabolismo , Dinorfinas/química , Dinorfinas/farmacologia , Células HEK293 , Humanos , Medições Luminescentes/métodos , Piperazinas/química , Piperazinas/farmacologia , Piperidinas/química , Piperidinas/farmacologia , Conformação Proteica , Pirrolidinas/química , Pirrolidinas/farmacologia , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anticorpos de Domínio Único/metabolismo , Tetra-Hidroisoquinolinas/química , Tetra-Hidroisoquinolinas/farmacologia
15.
Biochem J ; 477(7): 1203-1218, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32167135

RESUMO

Mutations in the Parkinson's disease (PD)-associated protein leucine-rich repeat kinase 2 (LRRK2) commonly lead to a reduction of GTPase activity and increase in kinase activity. Therefore, strategies for drug development have mainly been focusing on the design of LRRK2 kinase inhibitors. We recently showed that the central RocCOR domains (Roc: Ras of complex proteins; COR: C-terminal of Roc) of a bacterial LRRK2 homolog cycle between a dimeric and monomeric form concomitant with GTP binding and hydrolysis. PD-associated mutations can slow down GTP hydrolysis by stabilizing the protein in its dimeric form. Here, we report the identification of two Nanobodies (NbRoco1 and NbRoco2) that bind the bacterial Roco protein (CtRoco) in a conformation-specific way, with a preference for the GTP-bound state. NbRoco1 considerably increases the GTP turnover rate of CtRoco and reverts the decrease in GTPase activity caused by a PD-analogous mutation. We show that NbRoco1 exerts its effect by allosterically interfering with the CtRoco dimer-monomer cycle through the destabilization of the dimeric form. Hence, we provide the first proof of principle that allosteric modulation of the RocCOR dimer-monomer cycle can alter its GTPase activity, which might present a potential novel strategy to overcome the effect of LRRK2 PD mutations.


Assuntos
Proteínas de Bactérias/metabolismo , Chlorobi/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Domínios Proteicos , Anticorpos de Domínio Único/metabolismo , Proteínas ras/química , Regulação Alostérica , Animais , Camelídeos Americanos , Desenho de Fármacos , Escherichia coli/metabolismo , Hidrólise , Mutação , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/genética , Multimerização Proteica
16.
Curr Opin Struct Biol ; 60: 117-123, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32036243

RESUMO

Because of their small size and their beneficial biochemical and economic properties (size, affinity, specificity, stability, production cost), nanobodies are now increasingly used for routine and more innovative applications in research, biotechnology, and medicine. As they provide access to conformational epitopes in concave and hinge regions, nanobodies are also increasingly applied in structural biology to freeze dynamic proteins into single functional conformations. X-ray crystallography can then be used to determine the structures of different stills of the same moving biomolecule. Conformational nanobodies can also be introduced as intrabodies inside living cells as conformational biosensors for spatiotemporal analysis. By engineering these nanobodies in several ways, conformational nanobodies are now also amenable to single particle cryo-EM or to drive better-focused drug discovery.


Assuntos
Anticorpos de Domínio Único/química , Microscopia Crioeletrônica , Cristalografia por Raios X , Humanos , Conformação Proteica
17.
Elife ; 92020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31990273

RESUMO

Pentameric ligand-gated ion channels (pLGICs) or Cys-loop receptors are involved in fast synaptic signaling in the nervous system. Allosteric modulators bind to sites that are remote from the neurotransmitter binding site, but modify coupling of ligand binding to channel opening. In this study, we developed nanobodies (single domain antibodies), which are functionally active as allosteric modulators, and solved co-crystal structures of the prokaryote (Erwinia) channel ELIC bound either to a positive or a negative allosteric modulator. The allosteric nanobody binding sites partially overlap with those of small molecule modulators, including a vestibule binding site that is not accessible in some pLGICs. Using mutagenesis, we extrapolate the functional importance of the vestibule binding site to the human 5-HT3 receptor, suggesting a common mechanism of modulation in this protein and ELIC. Thus we identify key elements of allosteric binding sites, and extend drug design possibilities in pLGICs with an accessible vestibule site.


Assuntos
Proteínas de Bactérias , Erwinia/genética , Canais Iônicos de Abertura Ativada por Ligante , Receptores 5-HT3 de Serotonina , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Canais Iônicos de Abertura Ativada por Ligante/química , Canais Iônicos de Abertura Ativada por Ligante/genética , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Receptores 5-HT3 de Serotonina/química , Receptores 5-HT3 de Serotonina/genética , Receptores 5-HT3 de Serotonina/metabolismo , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/metabolismo
18.
PLoS Pathog ; 15(12): e1008139, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31815959

RESUMO

Prion or PrPSc is the proteinaceous infectious agent causing prion diseases in various mammalian species. Despite decades of research, the structural basis for PrPSc formation and prion infectivity remains elusive. To understand the role of the hydrophobic region in forming infectious prion at the molecular level, we report X-ray crystal structures of mouse (Mo) prion protein (PrP) (residues 89-230) in complex with a nanobody (Nb484). Using the recombinant prion propagation system, we show that the binding of Nb484 to the hydrophobic region of MoPrP efficiently inhibits the propagation of proteinase K resistant PrPSc and prion infectivity. In addition, when added to cultured mouse brain slices in high concentrations, Nb484 exhibits no neurotoxicity, which is drastically different from other neurotoxic anti-PrP antibodies, suggesting that the Nb484 can be a potential therapeutic agent against prion disease. In summary, our data provides the first structure-function evidence supporting a crucial role of the hydrophobic region of PrP in forming an infectious prion.


Assuntos
Proteínas PrPSc/química , Proteínas PrPSc/efeitos dos fármacos , Proteínas Priônicas/química , Proteínas Priônicas/efeitos dos fármacos , Anticorpos de Domínio Único/farmacologia , Animais , Camundongos , Conformação Proteica , Domínios Proteicos/efeitos dos fármacos , Relação Estrutura-Atividade
19.
Nat Chem Biol ; 15(12): 1156-1164, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31591563

RESUMO

Phospholipids are key components of cellular membranes and are emerging as important functional regulators of different membrane proteins, including pentameric ligand-gated ion channels (pLGICs). Here, we take advantage of the prokaryote channel ELIC (Erwinia ligand-gated ion channel) as a model to understand the determinants of phospholipid interactions in this family of receptors. A high-resolution structure of ELIC in a lipid-bound state reveals a phospholipid site at the lower half of pore-forming transmembrane helices M1 and M4 and at a nearby site for neurosteroids, cholesterol or general anesthetics. This site is shaped by an M4-helix kink and a Trp-Arg-Pro triad that is highly conserved in eukaryote GABAA/C and glycine receptors. A combined approach reveals that M4 is intrinsically flexible and that M4 deletions or disruptions of the lipid-binding site accelerate desensitization in ELIC, suggesting that lipid interactions shape the agonist response. Our data offer a structural context for understanding lipid modulation in pLGICs.


Assuntos
Ativação do Canal Iônico , Canais Iônicos/metabolismo , Lipídeos/química , Animais , Ligantes , Mutagênese , Xenopus
20.
Nature ; 573(7773): 287-290, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31485076

RESUMO

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 Viral
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