Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 171
Filtrar
1.
Nat Commun ; 13(1): 251, 2022 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-35017564

RESUMO

Influenza A viruses cause seasonal epidemics and global pandemics, representing a considerable burden to healthcare systems. Central to the replication cycle of influenza viruses is the viral RNA-dependent RNA polymerase which transcribes and replicates the viral RNA genome. The polymerase undergoes conformational rearrangements and interacts with viral and host proteins to perform these functions. Here we determine the structure of the 1918 influenza virus polymerase in transcriptase and replicase conformations using cryo-electron microscopy (cryo-EM). We then structurally and functionally characterise the binding of single-domain nanobodies to the polymerase of the 1918 pandemic influenza virus. Combining these functional and structural data we identify five sites on the polymerase which are sensitive to inhibition by nanobodies. We propose that the binding of nanobodies at these sites either prevents the polymerase from assuming particular functional conformations or interactions with viral or host factors. The polymerase is highly conserved across the influenza A subtypes, suggesting these sites as effective targets for potential influenza antiviral development.

2.
Mol Cell ; 2021 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-34890564

RESUMO

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.

3.
Proc Natl Acad Sci U S A ; 118(47)2021 11 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.

4.
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.

5.
Proc Natl Acad Sci U S A ; 118(33)2021 08 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 ; 29(12): 1371-1381.e6, 2021 Dec 02.
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.
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
8.
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.

9.
Molecules ; 26(12)2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34207949

RESUMO

BACKGROUND: Nanobodies, or VHHs, are derived from heavy chain-only antibodies (hcAbs) found in camelids. They overcome some of the inherent limitations of monoclonal antibodies (mAbs) and derivatives thereof, due to their smaller molecular size and higher stability, and thus present an alternative to mAbs for therapeutic use. Two nanobodies, Nb23 and Nb24, have been shown to similarly inhibit the self-aggregation of very amyloidogenic variants of ß2-microglobulin. Here, the structure of Nb23 was modeled with the Chemical-Shift (CS)-Rosetta server using chemical shift assignments from nuclear magnetic resonance (NMR) spectroscopy experiments, and used as prior knowledge in PONDEROSA restrained modeling based on experimentally assessed internuclear distances. Further validation was comparatively obtained with the results of molecular dynamics trajectories calculated from the resulting best energy-minimized Nb23 conformers. METHODS: 2D and 3D NMR spectroscopy experiments were carried out to determine the assignment of the backbone and side chain hydrogen, nitrogen and carbon resonances to extract chemical shifts and interproton separations for restrained modeling. RESULTS: The solution structure of isolated Nb23 nanobody was determined. CONCLUSIONS: The structural analysis indicated that isolated Nb23 has a dynamic CDR3 loop distributed over different orientations with respect to Nb24, which could determine differences in target antigen affinity or complex lability.


Assuntos
Anticorpos Monoclonais/química , Cadeias Pesadas de Imunoglobulinas/química , Espectroscopia de Ressonância Magnética/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Anticorpos de Domínio Único/química , Microglobulina beta-2/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/metabolismo , Elementos Estruturais de Proteínas , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/metabolismo , Microglobulina beta-2/imunologia
10.
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
11.
Prev Med ; 147: 106522, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33744328

RESUMO

Strategies to reduce dementia risk are needed to minimize the burden of this growing public health concern. Most individuals are not aware that dementia risk reduction is possible, let alone how this could be achieved. Health education, such as public awareness campaigns on the topic of dementia risk reduction, can meet this need. A public health campaign (including social media and offering an online individual risk assessment tool) was carried out over a 7-month period in Flanders, Belgium. Impact was assessed in two independent online surveys, before (n = 1003) and after the campaign (n = 1008), in representative samples of adults aged 40-75 years. Questions regarding personal needs, wishes and barriers were also included. After the campaign, more individuals (10.3%) were aware that dementia risk reduction is possible than before the campaign, and more individuals correctly identified 10 out of 12 surveyed modifiable dementia risk and protective factors. However, no differences were observed in low-educated individuals. Further, specific differences in potential needs, wishes and barriers for future campaigns or interventions were observed between demographic strata. The majority of the respondents (89%) indicated that they would welcome more information on improving their brain-health. More than half (54%) also believed that they lacked the necessary knowledge to make brain-healthy behavior changes. In conclusion, effective public awareness campaigns on the topic of dementia risk reduction are feasible and timely, given the state of the evidence. Special efforts need to be made to develop effective campaigns, tailored towards low-educated individuals.


Assuntos
Demência , Adulto , Conscientização , Bélgica , Demência/prevenção & controle , Conhecimentos, Atitudes e Prática em Saúde , Promoção da Saúde , Humanos , Comportamento de Redução do Risco
12.
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
13.
Aging Ment Health ; 25(8): 1376-1380, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-32590910

RESUMO

Many publications on dementia start by outlining the current estimated number of people with dementia and how that figure is going to double (in Western societies) or even quadruple (in developing countries) in the coming decades as a result of increasing life expectancy (in itself a good development). Dementia is therefore a huge challenge to society, both in terms of providing good care for persons living with dementia and their family caregivers, as well as in searching for curative solutions. Both these challenges are complex. Fortunately, recent research indicates primary prevention to be a promising additional strategy in the dementia quest. Now that epidemiological research robustly shows the link between lifestyle and risk of dementia, new challenges emerge, such as how to increase public awareness about brain health, how to develop and implement strategies to promote brain healthy lifestyles and how to avoid increasing health inequalities. Interdem, the pan-European network of researchers on Psychosocial Interventions in Dementia, strongly welcomes this new strategy and consequently established a taskforce on primary prevention. In this position paper, we outline what we see as main building blocks of primary prevention of dementia.


Assuntos
Demência , Cuidadores , Demência/epidemiologia , Demência/prevenção & controle , Humanos , Expectativa de Vida , Estilo de Vida , Prevenção Primária
14.
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
15.
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
16.
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
17.
EMBO Rep ; 21(12): e51761, 2020 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-33179844

RESUMO

Debates about the source of antibodies and their use are confusing two different issues. A ban on life immunization would have no repercussions on the quality of antibodies.


Assuntos
Anticorpos , Imunização , Animais
18.
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
20.
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
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...