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The transient receptor potential vanilloid 2 (TRPV2) ion channel is a polymodal receptor widely involved in many physiological and pathological processes. Despite many TRPV2 modulators being identified, whether and how TRPV2 is regulated by endogenous lipids remains elusive. Here, we report an endogenous cholesterol molecule inside the vanilloid binding pocket (VBP) of TRPV2, with a 'head down, tail up' configuration, resolved at 3.2 Å using cryo-EM. Cholesterol binding antagonizes ligand activation of TRPV2, which is removed from VBP by methyl-ß-cyclodextrin (MßCD) as resolved at 2.9 Å. We also observed that estradiol (E2) potentiated TRPV2 activation by 2-aminoethoxydiphenyl borate (2-APB), a classic tool compound for TRP channels. Our cryo-EM structures (resolved at 2.8-3.3 Å) further suggest how E2 disturbed cholesterol binding and how 2-APB bound within the VBP with E2 or without both E2 and endogenous cholesterol, respectively. Therefore, our study has established the structural basis for ligand recognition of the inhibitory endogenous cholesterol and excitatory exogenous 2-APB in TRPV2.
Assuntos
Canais de Cátion TRPV , Canais de Cátion TRPV/química , LigantesRESUMO
Continuum solvation models have been incredibly successful for the computationally efficient study of chemical reactions in solution. However, their development and application has generally been on focused on investigations of small, rigid molecules. Additional factors must be considered when studying large, flexible and multiply ionizable species. These include whether the use of thermocycle or entirely solution-phase approaches are more appropriate for the calculation of solution-phase free energies, which metrics can be used to reliably identify the conformation(s) adopted by flexible molecules in solution, and how errors due to inaccuracies in the prediction of low energy vibrational frequencies can be avoided. Here we explore these issues using the calculation of pKas for a diverse set of amine-containing species as a case study. We show that thermocycle-based approaches should only be applied where there are relatively small structural changes between the gas- and solution-phase molecular geometries, and that these methods are generally not appropriate for conformational searching. Using gas- or solution-phase energies or gas-phase free energies can also lead to errors in the identification of the most stable molecular conformation(s). Scaling of low energy vibrational modes (i.e., use of the quasi-harmonic oscillator approximation) is helpful, however care must be taken to ensure modes that change as part of the reaction are not disregarded. Entirely solution-phase approaches to the Gibbs free energy and hence pKa calculations were found to yield accurate pKa values for the amine test set studied when each charged site is complexed with an explicit water molecule and a proton exchange scheme is applied with an appropriately chosen reference acid.
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Tigecycline acts as a glycylcycline class bacteriostatic agent, and actively resists a series of bacteria, specifically drug fast bacteria. However, accumulating evidence showed that tetracycline and their derivatives such as doxycycline and minocycline have anti-cancer properties, which are out of their broader antimicrobial activity. We found that tigecycline dramatically inhibited gastric cancer cell proliferation and provided an evidence that tigecycline induced autophagy but not apoptosis in human gastric cancer cells. Further experiments demonstrated that AMPK pathway was activated accompanied with the suppression of its downstream targets including mTOR and p70S6K, and ultimately induced cell autophagy and inhibited cell growth. So our data suggested that tigecycline might act as a candidate agent for pre-clinical evaluation in treatment of patients suffering from gastric cancer.
Assuntos
Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Minociclina/análogos & derivados , Neoplasias Gástricas/tratamento farmacológico , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Camundongos , Camundongos SCID , Minociclina/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Serina-Treonina Quinases TOR/metabolismo , Tigeciclina , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The ClC-3 chloride/proton exchanger is both physiologically and pathologically critical, as it is potentiated by ATP to detect metabolic energy level and point mutations in ClC-3 lead to severe neurodegenerative diseases in human. However, why this exchanger is differentially modulated by ATP, ADP or AMP and how mutations caused gain-of-function remains largely unknow. Here we determine the high-resolution structures of dimeric wildtype ClC-3 in the apo state and in complex with ATP, ADP and AMP, and the disease-causing I607T mutant in the apo and ATP-bounded state by cryo-electron microscopy. In combination with patch-clamp recordings and molecular dynamic simulations, we reveal how the adenine nucleotides binds to ClC-3 and changes in ion occupancy between apo and ATP-bounded state. We further observe I607T mutation induced conformational changes and augments in current. Therefore, our study not only lays the structural basis of adenine nucleotides regulation in ClC-3, but also clearly indicates the target region for drug discovery against ClC-3 mediated neurodegenerative diseases.
Assuntos
Trifosfato de Adenosina , Canais de Cloreto , Microscopia Crioeletrônica , Simulação de Dinâmica Molecular , Doenças Neurodegenerativas , Canais de Cloreto/metabolismo , Canais de Cloreto/genética , Canais de Cloreto/química , Humanos , Trifosfato de Adenosina/metabolismo , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Nucleotídeos de Adenina/metabolismo , Técnicas de Patch-Clamp , Mutação , Difosfato de Adenosina/metabolismo , Células HEK293 , Monofosfato de Adenosina/metabolismo , Animais , Conformação ProteicaRESUMO
Antibodies against N-methyl-D-aspartate receptors (NMDARs) are most frequently detected in persons with autoimmune encephalitis (AE) and used as diagnostic biomarkers. Elucidating the structural basis of monoclonal antibody (mAb) binding to NMDARs would facilitate the development of targeted therapy for AE. Here, we reconstructed nanodiscs containing green fluorescent protein-fused NMDARs to label and sort individual immune B cells from persons with AE and further cloned and identified mAbs against NMDARs. This allowed cryo-electron microscopy analysis of NMDAR-Fab complexes, revealing that autoantibodies bind to the R1 lobe of the N-terminal domain of the GluN1 subunit. Small-angle X-ray scattering studies demonstrated NMDAR-mAb stoichiometry of 2:1 or 1:2, structurally suitable for mAb-induced clustering and endocytosis of NMDARs. Importantly, these mAbs reduced the surface NMDARs and NMDAR-mediated currents, without tonically affecting NMDAR channel gating. These structural and functional findings imply that the design of neutralizing antibody binding to the R1 lobe of NMDARs represents a potential therapy for AE treatment.
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The plasma membrane Na+/H+ exchanger Salt Overly Sensitive 1 (SOS1) is crucial for plant salt tolerance. Unlike typical sodium/proton exchangers, SOS1 contains a large cytoplasmic domain (CPD) that regulates Na+/H+ exchange activity. However, the underlying modulation mechanism remains unclear. Here we report the structures of SOS1 from Arabidopsis thaliana in two conformations, primarily differing in CPD flexibility. The CPD comprises an interfacial domain, a cyclic nucleotide-binding domain-like domain (CNBD-like domain) and an autoinhibition domain. Through yeast cell-based Na+ tolerance test, we reveal the regulatory role of the interfacial domain and the activation role of the CNBD-like domain. The CPD forms a negatively charged cavity that is connected to the ion binding site. The transport of Na+ may be coupled with the conformational change of CPD. These findings provide structural and functional insight into SOS1 activity regulation.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Tolerância ao Sal , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/metabolismoRESUMO
N-methyl-D-aspartate (NMDA) receptors are heterotetramers comprising two GluN1 and two alternate GluN2 (N2A-N2D) subunits. Here we report full-length cryo-EM structures of the human N1-N2D di-heterotetramer (di-receptor), rat N1-N2C di-receptor and N1-N2A-N2C tri-heterotetramer (tri-receptor) at a best resolution of 3.0 Å. The bilobate N-terminal domain (NTD) in N2D intrinsically adopts a closed conformation, leading to a compact NTD tetramer in the N1-N2D receptor. Additionally, crosslinking the ligand-binding domain (LBD) of two N1 protomers significantly elevated the channel open probability (Po) in N1-N2D di-receptors. Surprisingly, the N1-N2C di-receptor adopted both symmetric (minor) and asymmetric (major) conformations, the latter further locked by an allosteric potentiator, PYD-106, binding to a pocket between the NTD and LBD in only one N2C protomer. Finally, the N2A and N2C subunits in the N1-N2A-N2C tri-receptor display a conformation close to one protomer in the N1-N2A and N1-N2C di-receptors, respectively. These findings provide a comprehensive structural understanding of diverse function in major NMDA receptor subtypes.
Assuntos
Receptores de N-Metil-D-Aspartato , Ratos , Animais , Humanos , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Subunidades Proteicas/química , Domínios ProteicosRESUMO
Ion channels are expressed in almost all living cells, controlling the in-and-out communications, making them ideal drug targets, especially for central nervous system diseases. However, owing to their dynamic nature and the presence of a membrane environment, ion channels remain difficult targets for the past decades. Recent advancement in cryo-electron microscopy and computational methods has shed light on this issue. An explosion in high-resolution ion channel structures paved way for structure-based rational drug design and the state-of-the-art simulation and machine learning techniques dramatically improved the efficiency and effectiveness of computer-aided drug design. Here we present an overview of how simulation and machine learning-based methods fundamentally changed the ion channel-related drug design at different levels, as well as the emerging trends in the field.
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Polycystin-2 (PC2) is a transient receptor potential (TRP) channel present in ciliary membranes of the kidney. PC2 shares a transmembrane fold with other TRP channels, in addition to an extracellular domain found in TRPP and TRPML channels. Using molecular dynamics (MD) simulations and cryoelectron microscopy we identify and characterize PIP2 and cholesterol interactions with PC2. PC2 is revealed to have a PIP binding site close to the equivalent vanilloid/lipid binding site in the TRPV1 channel. A 3.0-Å structure reveals a binding site for cholesterol on PC2. Cholesterol interactions with the channel at this site are characterized by MD simulations. The two classes of lipid binding sites are compared with sites observed in other TRPs and in Kv channels. These findings suggest PC2, in common with other ion channels, may be modulated by both PIPs and cholesterol, and position PC2 within an emerging model of the roles of lipids in the regulation and organization of ciliary membranes.
Assuntos
Colesterol/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Canais de Cátion TRPP/química , Canais de Cátion TRPP/metabolismo , Animais , Sítios de Ligação , Microscopia Crioeletrônica , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Células Sf9RESUMO
Membranes in cells have defined distributions of lipids in each leaflet, controlled by lipid scramblases and flip/floppases. However, for some intracellular membranes such as the endoplasmic reticulum (ER) the scramblases have not been identified. Members of the TMEM16 family have either lipid scramblase or chloride channel activity. Although TMEM16K is widely distributed and associated with the neurological disorder autosomal recessive spinocerebellar ataxia type 10 (SCAR10), its location in cells, function and structure are largely uncharacterised. Here we show that TMEM16K is an ER-resident lipid scramblase with a requirement for short chain lipids and calcium for robust activity. Crystal structures of TMEM16K show a scramblase fold, with an open lipid transporting groove. Additional cryo-EM structures reveal extensive conformational changes from the cytoplasmic to the ER side of the membrane, giving a state with a closed lipid permeation pathway. Molecular dynamics simulations showed that the open-groove conformation is necessary for scramblase activity.