RESUMO
Hershey and Chase used bacteriophage T2 genome delivery inside Escherichia coli to demonstrate that DNA, not protein, is the genetic material. Seventy years later, our understanding of viral genome delivery in prokaryotes remains limited, especially for short-tailed phages of the Podoviridae family. These viruses expel mysterious ejection proteins found inside the capsid to form a DNA-ejectosome for genome delivery into bacteria. Here, we reconstitute the phage T7 DNA-ejectosome components gp14, gp15, and gp16 and solve the periplasmic tunnel structure at 2.7 Å resolution. We find that gp14 forms an outer membrane pore, gp15 assembles into a 210 Å hexameric DNA tube spanning the host periplasm, and gp16 extends into the host cytoplasm forming a â¼4,200 residue hub. Gp16 promotes gp15 oligomerization, coordinating peptidoglycan hydrolysis, DNA binding, and lipid insertion. The reconstituted gp15:gp16 complex lacks channel-forming activity, suggesting that the pore for DNA passage forms only transiently during genome ejection.
Assuntos
Bacteriófago T7/genética , DNA Viral/química , Periplasma/química , Proteínas do Core Viral/química , Biologia Computacional , Microscopia Crioeletrônica , Citoplasma/química , DNA Viral/metabolismo , Bicamadas Lipídicas/metabolismo , Periplasma/genética , Periplasma/metabolismo , Podoviridae/química , Podoviridae/genética , Proteínas do Core Viral/metabolismoRESUMO
Synaptic vesicle (SV) exo- and endocytosis are tightly coupled to sustain neurotransmission in presynaptic terminals, and both are regulated by Ca(2+). Ca(2+) influx triggered by voltage-gated Ca(2+) channels is necessary for SV fusion. However, extracellular Ca(2+) has also been shown to be required for endocytosis. The intracellular Ca(2+) levels (<1 microM) that trigger endocytosis are typically much lower than those (>10 microM) needed to induce exocytosis, and endocytosis is inhibited when the Ca(2+) level exceeds 1 microM. Here, we identify and characterize a transmembrane protein associated with SVs that, upon SV fusion, localizes at periactive zones. Loss of Flower results in impaired intracellular resting Ca(2+) levels and impaired endocytosis. Flower multimerizes and is able to form a channel to control Ca(2+) influx. We propose that Flower functions as a Ca(2+) channel to regulate synaptic endocytosis and hence couples exo- with endocytosis.
Assuntos
Canais de Cálcio/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Endocitose , Exocitose , Vesículas Sinápticas/metabolismo , Animais , Canais de Cálcio/análise , Proteínas de Drosophila/análise , Drosophila melanogaster/citologia , Isoformas de Proteínas/análise , Isoformas de Proteínas/metabolismo , Vesículas Sinápticas/químicaRESUMO
One approach to improve the utility of adeno-associated virus (AAV)-based gene therapy is to engineer the AAV capsid to (i) overcome poor transport through tissue barriers and (ii) redirect the broadly tropic AAV to disease-relevant cell types. Peptide- or protein-domain insertions into AAV surface loops can achieve both engineering goals by introducing a new interaction surface on the AAV capsid. However, we understand little about the impact of insertions on capsid structure and the extent to which engineered inserts depend on a specific capsid context to function. Here, we examine insert-capsid interactions for the engineered variant AAV9-PHP.B. The 7-amino-acid peptide insert in AAV9-PHP.B facilitates transport across the murine blood-brain barrier via binding to the receptor Ly6a. When transferred to AAV1, the engineered peptide does not bind Ly6a. Comparative structural analysis of AAV1-PHP.B and AAV9-PHP.B revealed that the inserted 7-amino-acid loop is highly flexible and has remarkably little impact on the surrounding capsid conformation. Our work demonstrates that Ly6a binding requires interactions with both the PHP.B peptide and specific residues from the AAV9 HVR VIII region. An AAV1-based vector that incorporates a larger region of AAV9-PHP.B-including the 7-amino-acid loop and adjacent HVR VIII amino acids-can bind to Ly6a and localize to brain tissue. However, unlike AAV9-PHP.B, this AAV1-based vector does not penetrate the blood-brain barrier. Here we discuss the implications for AAV capsid engineering and the transfer of engineered activities between serotypes. IMPORTANCE Targeting AAV vectors to specific cellular receptors is a promising strategy for enhancing expression in target cells or tissues while reducing off-target transgene expression. The AAV9-PHP.B/Ly6a interaction provides a model system with a robust biological readout that can be interrogated to better understand the biology of AAV vectors' interactions with target receptors. In this work, we analyzed the sequence and structural features required to successfully transfer the Ly6a receptor-binding epitope from AAV9-PHP.B to another capsid of clinical interest, AAV1. We found that AAV1- and AAV9-based vectors targeted to the same receptor exhibited different brain-transduction profiles. Our work suggests that, in addition to attachment-receptor binding, the capsid context in which this binding occurs is important for a vector's performance.
Assuntos
Terapia Genética/métodos , Vetores Genéticos/genética , Ligação Proteica/genética , Aminoácidos/genética , Animais , Antígenos Ly/metabolismo , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Dependovirus/genética , Dependovirus/metabolismo , Técnicas de Transferência de Genes , Engenharia Genética/métodos , Células HEK293 , Humanos , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Peptídeos/genética , Domínios Proteicos/genética , Transdução Genética/métodos , Transgenes/genéticaRESUMO
Thermosensitive transient receptor potential (TRP) ion channels detect changes in ambient temperature to regulate body temperature and temperature-dependent cellular activity. Rodent orthologs of TRP vanilloid 2 (TRPV2) are activated by nonphysiological heat exceeding 50 °C, and human TRPV2 is heat-insensitive. TRPV2 is required for phagocytic activity of macrophages which are rarely exposed to excessive heat, but what activates TRPV2 in vivo remains elusive. Here we describe the molecular mechanism of an oxidation-induced temperature-dependent gating of TRPV2. While high concentrations of H2O2 induce a modest sensitization of heat-induced inward currents, the oxidant chloramine-T (ChT), ultraviolet A light, and photosensitizing agents producing reactive oxygen species (ROS) activate and sensitize TRPV2. This oxidation-induced activation also occurs in excised inside-out membrane patches, indicating a direct effect on TRPV2. The reducing agent dithiothreitol (DTT) in combination with methionine sulfoxide reductase partially reverses ChT-induced sensitization, and the substitution of the methionine (M) residues M528 and M607 to isoleucine almost abolishes oxidation-induced gating of rat TRPV2. Mass spectrometry on purified rat TRPV2 protein confirms oxidation of these residues. Finally, macrophages generate TRPV2-like heat-induced inward currents upon oxidation and exhibit reduced phagocytosis when exposed to the TRP channel inhibitor ruthenium red (RR) or to DTT. In summary, our data reveal a methionine-dependent redox sensitivity of TRPV2 which may be an important endogenous mechanism for regulation of TRPV2 activity and account for its pivotal role for phagocytosis in macrophages.
Assuntos
Metionina/metabolismo , Canais de Cátion TRPV/química , Canais de Cátion TRPV/metabolismo , Canais de Cálcio/química , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Cloraminas/química , Escherichia coli/genética , Temperatura Alta , Humanos , Peróxido de Hidrogênio/química , Macrófagos , Metionina/química , Mutação , Oxidantes/química , Oxirredução , Técnicas de Patch-Clamp , Fagocitose , Canais de Cátion TRPM/química , Canais de Cátion TRPM/metabolismo , Canais de Cátion TRPV/genética , Compostos de Tosil/químicaRESUMO
Transient Receptor Potential (TRP) channels are evolutionarily conserved integral membrane proteins. The mammalian TRP superfamily of ion channels consists of 28 cation permeable channels that are grouped into six subfamilies based on sequence homology (Fig. 6.1). The canonical TRP (TRPC) subfamily is known for containing the founding member of mammalian TRP channels. The vanilloid TRP (TRPV) subfamily has been extensively studied due to the heat sensitivity of its founding member. The melastatin-related TRP (TRPM) subfamily includes some of the few known bi-functional ion channels, which contain functional enzymatic domains. The ankyrin TRP (TRPA) subfamily consists of a single chemo-nociceptor that has been proposed to be a target for analgesics. The mucolipin TRP (TRPML) subfamily channels are found primarily in intracellular compartments and were discovered based on their critical role in type IV mucolipidosis (ML-IV). The polycystic TRP (TRPP) subfamily is a diverse group of proteins implicated in autosomal dominant polycystic kidney disease (ADPKD). Overall, this superfamily of channels is involved in a vast array of physiological and pathophysiological processes making the study of these channels imperative to our understanding of subcellular biochemistry.
Assuntos
Mucolipidoses , Família Multigênica , Rim Policístico Autossômico Dominante , Canais de Potencial de Receptor Transitório , Animais , Humanos , Mucolipidoses/genética , Mucolipidoses/metabolismo , Mucolipidoses/patologia , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Domínios Proteicos , Homologia de Sequência de Aminoácidos , Canais de Potencial de Receptor Transitório/química , Canais de Potencial de Receptor Transitório/genética , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
The esterification of alcohols with fatty acids is a universal mechanism to form inert storage forms of sterols, di- and triacylglycerols, and retinoids. In ocular tissues, formation of retinyl esters is an essential step in the enzymatic regeneration of the visual chromophore (11-cis-retinal). Acyl-CoA wax alcohol acyltransferase 2 (AWAT2), also known as multifunctional O-acyltransferase (MFAT), is an integral membrane enzyme with a broad substrate specificity that has been shown to preferentially esterify 11-cis-retinol and thus contribute to formation of a readily available pool of cis retinoids in the eye. However, the mechanism by which this promiscuous enzyme can gain substrate specificity is unknown. Here, we provide evidence for an allosteric modulation of the enzymatic activity by 11-cis retinoids. This regulation is independent from cellular retinaldehyde-binding protein (CRALBP), the major cis-retinoid binding protein. This positive-feedback regulation leads to decreased esterification rates for 9-cis, 13-cis, or all-trans retinols and thus enables preferential synthesis of 11-cis-retinyl esters. Finally, electron microscopy analyses of the purified enzyme indicate that this allosteric effect does not result from formation of functional oligomers. Altogether, these data provide the experimental basis for understanding regulation of AWAT2 substrate specificity.
Assuntos
Aciltransferases/metabolismo , Proteínas de Transporte/genética , Olho/metabolismo , Retinoides/metabolismo , Vitamina A/metabolismo , Aciltransferases/química , Álcoois/metabolismo , Regulação Alostérica/genética , Animais , Proteínas de Transporte/metabolismo , Esterificação , Ésteres/metabolismo , Olho/crescimento & desenvolvimento , Olho/ultraestrutura , Ácidos Graxos/metabolismo , Humanos , Camundongos , Microscopia Eletrônica , Retinoides/genética , Especificidade por Substrato , Vitamina A/biossínteseRESUMO
Amphipathic polymers (amphipols), such as A8-35 and SApol, are a new tool for stabilizing integral membrane proteins in detergent-free conditions for structural and functional studies. Transient receptor potential (TRP) ion channels function as tetrameric protein complexes in a diverse range of cellular processes including sensory transduction. Mammalian TRP channels share ~20 % sequence similarity and are categorized into six subfamilies: TRPC (canonical), TRPV (vanilloid), TRPA (ankyrin), TRPM (melastatin), TRPP (polycystin), and TRPML (mucolipin). Due to the inherent difficulties in purifying eukaryotic membrane proteins, structural studies of TRP channels have been limited. Recently, A8-35 was essential in resolving the molecular architecture of the nociceptor TRPA1 and led to the determination of a high-resolution structure of the thermosensitive TRPV1 channel by cryo-EM. Newly developed maltose-neopentyl glycol (MNG) detergents have also proven to be useful in stabilizing TRP channels for structural analysis. In this review, we will discuss the impacts of amphipols and MNG detergents on structural studies of TRP channels by cryo-EM. We will compare how A8-35 and MNG detergents interact with the hydrophobic transmembrane domains of TRP channels. In addition, we will discuss what these cryo-EM studies reveal on the importance of screening different types of surfactants toward determining high-resolution structures of TRP channels.
Assuntos
Membrana Celular/química , Membrana Celular/metabolismo , Polímeros/química , Propilaminas/química , Tensoativos/química , Canais de Potencial de Receptor Transitório/química , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Humanos , Interações Hidrofóbicas e Hidrofílicas , Solubilidade , Relação Estrutura-Atividade , Canais de Potencial de Receptor Transitório/ultraestruturaRESUMO
Temperature sensation is important for adaptation and survival of organisms. While temperature has the potential to affect all biological macromolecules, organisms have evolved specific thermosensitive molecular detectors that are able to transduce temperature changes into physiologically relevant signals. Among these thermosensors are ion channels from the transient receptor potential (TRP) family. Prime candidates include TRPV1-4, TRPA1, and TRPM8 (the so-called "thermoTRP" channels), which are expressed in sensory neurons and gated at specific temperatures. Electrophysiological and thermodynamic approaches have been employed to determine the nature by which thermoTRPs detect temperature and couple temperature changes to channel gating. To further understand how thermoTRPs sense temperature, high-resolution structures of full-length thermoTRPs channels will be required. Here, we will discuss current progress in unraveling the structures of thermoTRP channels.
Assuntos
Sensação Térmica , Canais de Potencial de Receptor Transitório/química , Canais de Potencial de Receptor Transitório/metabolismo , Animais , HumanosRESUMO
The calcium-selective TRPV5 channel activated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is involved in calcium homeostasis. Recently, cryoelectron microscopy (cryo-EM) provided molecular details of TRPV5 modulation by exogenous and endogenous molecules. However, the details of TRPV5 inhibition by the antifungal agent econazole (ECN) remain elusive due to the low resolution of the currently available structure. In this study, we employ cryo-EM to comprehensively examine how the ECN inhibits TRPV5. By combining our structural findings with site-directed mutagenesis, calcium measurements, electrophysiology, and molecular dynamics simulations, we determined that residues F472 and L475 on the S4 helix, along with residue W495 on the S5 helix, collectively constitute the ECN-binding site. Additionally, the structure of TRPV5 in the presence of ECN and PI(4,5)P2, which does not show the bound activator, reveals a potential inhibition mechanism in which ECN competes with PI(4,5)P2, preventing the latter from binding, and ultimately pore closure.
Assuntos
Antifúngicos , Econazol , Canais de Cátion TRPV , Antifúngicos/farmacologia , Cálcio/metabolismo , Microscopia Crioeletrônica , Econazol/farmacologia , Simulação de Dinâmica Molecular , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/químicaRESUMO
TRPA1 (transient receptor potential ankyrin 1) is an ion channel expressed in the termini of sensory neurons and is activated in response to a broad array of noxious exogenous and endogenous thiol-reactive compounds, making it a crucial player in chemical nociception. A number of conserved cysteine residues on the N-terminal domain of the channel have been identified as critical for sensing these electrophilic pungent chemicals, and our recent EM structure with modeled domains predicts that these cysteines form a ligand-binding pocket, allowing for the possibility of disulfide bonding between the cysteine residues. Here, we present a comprehensive mass spectrometry investigation of the in vivo disulfide bonding conformation and in vitro reactivity of 30 of the 31 cysteine residues in the TRPA1 ion channel. Four disulfide bonds were detected in the in vivo TRPA1 structure: Cys-666-Cys-622, Cys-666-Cys-463, Cys-622-Cys-609, and Cys-666-Cys-193. All of the cysteines detected were reactive to N-methylmaleimide (NMM) in vitro, with varying degrees of labeling efficiency. Comparison of the ratio of the labeling efficiency at 300 µM versus 2 mM NMM identified a number of cysteine residues that were outliers from the mean labeling ratio, suggesting that protein conformation changes rendered these cysteines either more or less protected from labeling at the higher NMM concentrations. These results indicate that the activation mechanism of TRPA1 may involve N-terminal conformation changes and disulfide bonding between critical cysteine residues.
Assuntos
Dissulfetos/química , Ativação do Canal Iônico/fisiologia , Canais de Potencial de Receptor Transitório/química , Canais de Potencial de Receptor Transitório/fisiologia , Animais , Cisteína/química , Espectrometria de Massas , Proteínas de Membrana/química , Proteínas de Membrana/fisiologia , Camundongos , Nociceptores/fisiologia , Conformação Proteica , Saccharomyces cerevisiae , Células Receptoras Sensoriais/fisiologia , Compostos de Sulfidrila/química , Canal de Cátion TRPA1RESUMO
Transient receptor potential vanilloid (TRPV) channels play various important roles in human physiology. As membrane proteins, these channels are modulated by their endogenous lipid environment as the recent wealth of structural studies has revealed functional and structural lipid binding sites. Additionally, it has been shown that exogenous ligands can exchange with some of these lipids to alter channel gating. Here, we used molecular dynamics simulations to examine how one member of the TRPV family, TRPV2, interacts with endogenous lipids and the pharmacological modulator cannabidiol (CBD). By computationally reconstituting TRPV2 into a typical plasma membrane environment, which includes phospholipids, cholesterol, and phosphatidylinositol (PIP) in the inner leaflet, we showed that most of the interacting surface lipids are phospholipids without strong specificity for headgroup types. Intriguingly, we observed that the C-terminal membrane proximal region of the channel binds preferentially to PIP lipids. We also modelled two structural lipids in the simulation: one in the vanilloid pocket and the other in the voltage sensor-like domain (VSLD) pocket. The simulation shows that the VSLD lipid dampens the fluctuation of the VSLD residues, while the vanilloid lipid exhibits heterogeneity both in its binding pose and in its influence on protein dynamics. Addition of CBD to our simulation system led to an open selectivity filter and a structural rearrangement that includes a clockwise rotation of the ankyrin repeat domains, TRP helix, and VSLD. Together, these results reveal the interplay between endogenous lipids and an exogenous ligand and their effect on TRPV2 stability and channel gating.
Assuntos
Antineoplásicos , Canais de Cátion TRPV , Humanos , Canais de Cátion TRPV/química , Ligantes , Repetição de Anquirina , Sítios de Ligação , FosfolipídeosRESUMO
Transient receptor potential (TRP) ion channels are gated by diverse intra- and extracellular stimuli leading to cation inflow (Na+, Ca2+) regulating many cellular processes and initiating organismic somatosensation. Structures of most TRP channels have been solved. However, structural and sequence analysis showed that ~30% of the TRP channel sequences, mainly the N- and C-termini, are intrinsically disordered regions (IDRs). Unfortunately, very little is known about IDR 'structure', dynamics and function, though it has been shown that they are essential for native channel function. Here, we imaged TRPV2 channels in membranes using high-speed atomic force microscopy (HS-AFM). The dynamic single molecule imaging capability of HS-AFM allowed us to visualize IDRs and revealed that N-terminal IDRs were involved in intermolecular interactions. Our work provides evidence about the 'structure' of the TRPV2 IDRs, and that the IDRs may mediate protein-protein interactions.
Assuntos
Proteínas Intrinsicamente Desordenadas , Canais de Cátion TRPV , Microscopia de Força Atômica , Imagem Individual de MoléculaRESUMO
Transient receptor potential ankyrin 1 (TRPA1) is a non-selective ion channel, which is expressed in nociceptor sensory neurons and transduces chemical, inflammatory, and neuropathic pain signals. Numerous non-reactive compounds and electrophilic compounds, such as endogenous inflammatory mediators and exogenous pungent chemicals, can activate TRPA1. Here we report a 16-Å resolution structure of purified, functional, amphipol-stabilized TRPA1 analyzed by single-particle EM. Molecular models of the N and C termini of the channel were generated using the I-TASSER protein structure prediction server and docked into the EM density to provide insight into the TRPA1 subunit organization. This structural analysis suggests a location for critical N-terminal cysteine residues involved in electrophilic activation at the interface between neighboring subunits. Our results indicate that covalent modifications within this pocket may alter interactions between subunits and promote conformational changes that lead to channel activation.
Assuntos
Canais de Potencial de Receptor Transitório/ultraestrutura , Animais , Cálcio/química , Cromatografia em Gel , Dicroísmo Circular , Inflamação , Ligantes , Camundongos , Microscopia Eletrônica/métodos , Modelos Moleculares , Conformação Molecular , Conformação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade , Canal de Cátion TRPA1 , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
Transient Receptor Potential Vanilloid 5 and 6 (TRPV5 and TRPV6) are Ca2+ selective epithelial ion channels. They are the products of a relatively recent gene duplication in mammals, and have high sequence homology to each other. Their functional properties are also much more similar to each other than to other members of the TRPV subfamily. They are both constitutively active, and this activity depends on the endogenous cofactor phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Both channels undergo Ca2+-induced inactivation, which is mediated by direct binding of the ubiquitous Ca2+ binding protein calmodulin (CaM) to the channels, and by a decrease in PI(4,5)P2 levels by Ca2+ -induced activation of phospholipase C (PLC). Recent cryo electron microscopy (cryo-EM) and X-ray crystallography structures provided detailed structural information for both TRPV5 and TRPV6. This review will discuss this structural information in the context of the function of these channels focusing on the mechanism of CaM inhibition, activation by PI(4,5)P2 and binding of pharmacological modulators.
Assuntos
Cálcio , Canais de Cátion TRPV , Animais , Cálcio/metabolismo , Calmodulina/metabolismo , Microscopia Crioeletrônica , Mamíferos/metabolismo , Fosfatidilinositóis , Canais de Cátion TRPV/metabolismoRESUMO
Transient receptor potential vanilloid 5 (TRPV5) is a kidney-specific Ca2+-selective ion channel that plays a key role in Ca2+ homeostasis. The basal activity of TRPV5 is balanced through activation by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and inhibition by Ca2+-bound calmodulin (CaM). Parathyroid hormone (PTH), the key extrinsic regulator of Ca2+ homeostasis, increases the activity of TRPV5 via protein kinase A (PKA)-mediated phosphorylation. Metabolic acidosis leads to reduced TRPV5 activity independent of PTH, causing hypercalciuria. Using cryoelectron microscopy (cryo-EM), we show that low pH inhibits TRPV5 by precluding PI(4,5)P2 activation. We capture intermediate conformations at low pH, revealing a transition from open to closed state. In addition, we demonstrate that PI(4,5)P2 is the primary modulator of channel gating, yet PKA controls TRPV5 activity by preventing CaM binding and channel inactivation. Our data provide detailed molecular mechanisms for regulation of TRPV5 by two key extrinsic modulators, low pH and PKA.
Assuntos
Cálcio , Canais de Cátion TRPV , Cálcio/metabolismo , Sinalização do Cálcio , Calmodulina/metabolismo , Microscopia Crioeletrônica , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Hormônio Paratireóideo , Canais de Cátion TRPV/genéticaRESUMO
Transient receptor potential (TRP) channels emerged in fungi as mechanosensitive osmoregulators. The Saccharomyces cerevisiae vacuolar TRP yeast 1 (TRPY1) is the most studied TRP channel from fungi, but the structure and details of channel modulation remain elusive. Here, we describe the full-length cryoelectron microscopy structure of TRPY1 at 3.1 Å resolution in a closed state. The structure, despite containing an evolutionarily conserved and archetypical transmembrane domain, reveals distinctive structural folds for the cytosolic N and C termini, compared with other eukaryotic TRP channels. We identify an inhibitory phosphatidylinositol 3-phosphate (PI(3)P) lipid-binding site, along with two Ca2+-binding sites: a cytosolic site, implicated in channel activation and a vacuolar lumen site, implicated in inhibition. These findings, together with data from microsecond-long molecular dynamics simulations and a model of a TRPY1 open state, provide insights into the basis of TRPY1 channel modulation by lipids and Ca2+, and the molecular evolution of TRP channels.
Assuntos
Cálcio/metabolismo , Fosfatos de Fosfatidilinositol/farmacologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Canais de Cátion TRPC/química , Canais de Cátion TRPC/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Citosol/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Fosfatos de Fosfatidilinositol/química , Conformação Proteica , Domínios Proteicos , Multimerização Proteica , Saccharomyces cerevisiae/químicaRESUMO
Transient receptor potential vanilloid 2 (TRPV2) is involved in many critical physiological and pathophysiological processes, making it a promising drug target. Here we present cryo-electron microscopy (cryo-EM) structures of rat TRPV2 in lipid nanodiscs activated by 2-aminoethoxydiphenyl borate (2-APB) and propose a TRPV2-specific 2-ABP binding site at the interface of S5 of one monomer and the S4-S5 linker of the adjacent monomer. In silico docking and electrophysiological studies confirm the key role of His521 and Arg539 in 2-APB activation of TRPV2. Additionally, electrophysiological experiments show that the combination of 2-APB and cannabidiol has a synergetic effect on TRPV2 activation, and cryo-EM structures demonstrate that both drugs were able to bind simultaneously. Together, our cryo-EM structures represent multiple functional states of the channel, providing a native picture of TRPV2 activation by small molecules and a structural framework for the development of TRPV2-specific activators.
Assuntos
Canais de Cátion TRPV , Animais , Sítios de Ligação , Microscopia Crioeletrônica , Domínios Proteicos , Ratos , Canais de Cátion TRPV/metabolismoRESUMO
The transient receptor potential (TRP) family of ion channels participate in many signaling pathways. TRPV1 functions as a molecular integrator of noxious stimuli, including heat, low pH, and chemical ligands. Here, we report the 3D structure of full-length rat TRPV1 channel expressed in the yeast Saccharomyces cerevisiae and purified by immunoaffinity chromatography. We demonstrate that the recombinant purified TRPV1 channel retains its structural and functional integrity and is suitable for structural analysis. The 19-A structure of TRPV1 determined by using single-particle electron cryomicroscopy exhibits fourfold symmetry and comprises two distinct regions: a large open basket-like domain, likely corresponding to the cytoplasmic N- and C-terminal portions, and a more compact domain, corresponding to the transmembrane portion. The assignment of transmembrane and cytoplasmic regions was supported by fitting crystal structures of the structurally homologous Kv1.2 channel and isolated TRPV1 ankyrin repeats into the TRPV1 structure.
Assuntos
Canais de Cátion TRPV/química , Animais , Repetição de Anquirina , Membrana Celular/química , Microscopia Crioeletrônica/métodos , Cristalografia , Citoplasma/química , Imageamento Tridimensional , Canal de Potássio Kv1.2/química , Conformação Proteica , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Saccharomyces cerevisiae/genética , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/isolamento & purificaçãoRESUMO
The transient receptor potential (TRP) vanilloid 2 (TRPV2) and TRP vanilloid 5 (TRPV5) cation channels play an important role in various physiological and pathophysiological processes. The heterologous expression and purification of these channels is critical for functional and structural characterization of these important proteins. Full-length rat TRPV2 and rabbit TRPV5 can both be expressed in Saccharomyces cerevisiae and affinity purified using the 1D4 epitope and antibody to yield pure, functional channels. Further, these channels can be reconstituted into lipid nanodiscs for a more functionally relevant environment. Presented here are protocols for the expression of full-length rat TRPV2 and rabbit TRPV5 in Saccharomyces cerevisiae, their affinity purification, and their reconstitution into nanodiscs for structural and functional studies.
Assuntos
Canais de Cátion TRPV , Animais , Coelhos , Ratos , Canais de Cátion TRPV/genéticaRESUMO
Transient receptor potential (TRP) ion channels are a super-family of ion channels that mediate transmembrane cation flux with polymodal activation, ranging from chemical to physical stimuli. Furthermore, due to their ubiquitous expression and role in human diseases, they serve as potential pharmacological targets. Advances in cryo-EM TRP channel structural biology has revealed general, as well as diverse, architectural elements and regulatory sites among TRP channel subfamilies. Here, we review the endogenous and pharmacological ligand-binding sites of TRP channels and their regulatory mechanisms.