RESUMO
KCR channelrhodopsins (K+-selective light-gated ion channels) have received attention as potential inhibitory optogenetic tools but more broadly pose a fundamental mystery regarding how their K+ selectivity is achieved. Here, we present 2.5-2.7 Å cryo-electron microscopy structures of HcKCR1 and HcKCR2 and of a structure-guided mutant with enhanced K+ selectivity. Structural, electrophysiological, computational, spectroscopic, and biochemical analyses reveal a distinctive mechanism for K+ selectivity; rather than forming the symmetrical filter of canonical K+ channels achieving both selectivity and dehydration, instead, three extracellular-vestibule residues within each monomer form a flexible asymmetric selectivity gate, while a distinct dehydration pathway extends intracellularly. Structural comparisons reveal a retinal-binding pocket that induces retinal rotation (accounting for HcKCR1/HcKCR2 spectral differences), and design of corresponding KCR variants with increased K+ selectivity (KALI-1/KALI-2) provides key advantages for optogenetic inhibition in vitro and in vivo. Thus, discovery of a mechanism for ion-channel K+ selectivity also provides a framework for next-generation optogenetics.
Assuntos
Channelrhodopsins , Rhinosporidium , Humanos , Channelrhodopsins/química , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Channelrhodopsins/ultraestrutura , Microscopia Crioeletrônica , Canais Iônicos , Potássio/metabolismo , Rhinosporidium/químicaRESUMO
ChRmine, a recently discovered pump-like cation-conducting channelrhodopsin, exhibits puzzling properties (large photocurrents, red-shifted spectrum, and extreme light sensitivity) that have created new opportunities in optogenetics. ChRmine and its homologs function as ion channels but, by primary sequence, more closely resemble ion pump rhodopsins; mechanisms for passive channel conduction in this family have remained mysterious. Here, we present the 2.0 Å resolution cryo-EM structure of ChRmine, revealing architectural features atypical for channelrhodopsins: trimeric assembly, a short transmembrane-helix 3, a twisting extracellular-loop 1, large vestibules within the monomer, and an opening at the trimer interface. We applied this structure to design three proteins (rsChRmine and hsChRmine, conferring further red-shifted and high-speed properties, respectively, and frChRmine, combining faster and more red-shifted performance) suitable for fundamental neuroscience opportunities. These results illuminate the conduction and gating of pump-like channelrhodopsins and point the way toward further structure-guided creation of channelrhodopsins for applications across biology.
Assuntos
Channelrhodopsins/química , Channelrhodopsins/metabolismo , Ativação do Canal Iônico , Animais , Channelrhodopsins/ultraestrutura , Microscopia Crioeletrônica , Feminino , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Modelos Moleculares , Optogenética , Filogenia , Ratos Sprague-Dawley , Bases de Schiff/química , Células Sf9 , Relação Estrutura-AtividadeRESUMO
Cannabis elicits its mood-enhancing and analgesic effects through the cannabinoid receptor 1 (CB1), a G protein-coupled receptor (GPCR) that signals primarily through the adenylyl cyclase-inhibiting heterotrimeric G protein Gi. Activation of CB1-Gi signaling pathways holds potential for treating a number of neurological disorders and is thus crucial to understand the mechanism of Gi activation by CB1. Here, we present the structure of the CB1-Gi signaling complex bound to the highly potent agonist MDMB-Fubinaca (FUB), a recently emerged illicit synthetic cannabinoid infused in street drugs that have been associated with numerous overdoses and fatalities. The structure illustrates how FUB stabilizes the receptor in an active state to facilitate nucleotide exchange in Gi. The results compose the structural framework to explain CB1 activation by different classes of ligands and provide insights into the G protein coupling and selectivity mechanisms adopted by the receptor.
Assuntos
Receptor CB1 de Canabinoide/metabolismo , Receptor CB1 de Canabinoide/ultraestrutura , Animais , Agonistas de Receptores de Canabinoides/farmacologia , Canabinoides/farmacologia , Microscopia Crioeletrônica/métodos , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Indazóis/farmacologia , Ligantes , Ligação Proteica , Receptor CB1 de Canabinoide/química , Receptores de Canabinoides/química , Receptores de Canabinoides/metabolismo , Receptores de Canabinoides/ultraestrutura , Receptores Acoplados a Proteínas G/metabolismo , Células Sf9 , Transdução de Sinais/efeitos dos fármacosRESUMO
Endogenous parathyroid hormone (PTH) and PTH-related peptide (PTHrP) bind to the parathyroid hormone receptor 1 (PTH1R) and activate the stimulatory G-protein (Gs) signaling pathway. Intriguingly, the two ligands have distinct signaling and physiological properties: PTH evokes prolonged Gs activation, whereas PTHrP evokes transient Gs activation with reduced bone-resorption effects. The distinct molecular actions are ascribed to the differences in ligand recognition and dissociation kinetics. Here, we report cryoelectron microscopic structures of six forms of the human PTH1R-Gs complex in the presence of PTH or PTHrP at resolutions of 2.8 -4.1 Å. A comparison of the PTH-bound and PTHrP-bound structures reveals distinct ligand-receptor interactions underlying the ligand affinity and selectivity. Furthermore, five distinct PTH-bound structures, combined with computational analyses, provide insights into the unique and complex process of ligand dissociation from the receptor and shed light on the distinct durations of signaling induced by PTH and PTHrP.
Assuntos
Proteína Relacionada ao Hormônio Paratireóideo , Receptor Tipo 1 de Hormônio Paratireóideo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Humanos , Ligantes , Hormônio Paratireóideo/química , Hormônio Paratireóideo/metabolismo , Hormônio Paratireóideo/farmacologia , Proteína Relacionada ao Hormônio Paratireóideo/química , Proteína Relacionada ao Hormônio Paratireóideo/genética , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Receptor Tipo 1 de Hormônio Paratireóideo/genética , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismoRESUMO
Arrestin proteins bind to active, phosphorylated G-protein-coupled receptors (GPCRs), thereby preventing G-protein coupling, triggering receptor internalization and affecting various downstream signalling pathways1,2. Although there is a wealth of structural information detailing the interactions between GPCRs and G proteins, less is known about how arrestins engage GPCRs. Here we report a cryo-electron microscopy structure of full-length human neurotensin receptor 1 (NTSR1) in complex with truncated human ß-arrestin 1 (ßarr1(ΔCT)). We find that phosphorylation of NTSR1 is critical for the formation of a stable complex with ßarr1(ΔCT), and identify phosphorylated sites in both the third intracellular loop and the C terminus that may promote this interaction. In addition, we observe a phosphatidylinositol-4,5-bisphosphate molecule forming a bridge between the membrane side of NTSR1 transmembrane segments 1 and 4 and the C-lobe of arrestin. Compared with a structure of a rhodopsin-arrestin-1 complex, in our structure arrestin is rotated by approximately 85° relative to the receptor. These findings highlight both conserved aspects and plasticity among arrestin-receptor interactions.
Assuntos
Modelos Moleculares , Receptores de Neurotensina/química , beta-Arrestina 1/química , Microscopia Crioeletrônica , Humanos , Fosforilação , Estabilidade Proteica , Estrutura Quaternária de Proteína , Receptores de Neurotensina/metabolismo , beta-Arrestina 1/metabolismoRESUMO
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Neurotensin receptor 1 (NTSR1) is a G-protein-coupled receptor (GPCR) that engages multiple subtypes of G protein, and is involved in the regulation of blood pressure, body temperature, weight and the response to pain. Here we present structures of human NTSR1 in complex with the agonist JMV449 and the heterotrimeric Gi1 protein, at a resolution of 3 Å. We identify two conformations: a canonical-state complex that is similar to recently reported GPCR-Gi/o complexes (in which the nucleotide-binding pocket adopts more flexible conformations that may facilitate nucleotide exchange), and a non-canonical state in which the G protein is rotated by about 45 degrees relative to the receptor and exhibits a more rigid nucleotide-binding pocket. In the non-canonical state, NTSR1 exhibits features of both active and inactive conformations, which suggests that the structure may represent an intermediate form along the activation pathway of G proteins. This structural information, complemented by molecular dynamics simulations and functional studies, provides insights into the complex process of G-protein activation.
Assuntos
Microscopia Crioeletrônica , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/ultraestrutura , Receptores de Neurotensina/química , Receptores de Neurotensina/ultraestrutura , Sítios de Ligação , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Ligação Proteica , Conformação Proteica , Receptores de Neurotensina/agonistas , Receptores de Neurotensina/metabolismoRESUMO
The naturally occurring channelrhodopsin variant anion channelrhodopsin-1 (ACR1), discovered in the cryptophyte algae Guillardia theta, exhibits large light-gated anion conductance and high anion selectivity when expressed in heterologous settings, properties that support its use as an optogenetic tool to inhibit neuronal firing with light. However, molecular insight into ACR1 is lacking owing to the absence of structural information underlying light-gated anion conductance. Here we present the crystal structure of G. theta ACR1 at 2.9 Å resolution. The structure reveals unusual architectural features that span the extracellular domain, retinal-binding pocket, Schiff-base region, and anion-conduction pathway. Together with electrophysiological and spectroscopic analyses, these findings reveal the fundamental molecular basis of naturally occurring light-gated anion conductance, and provide a framework for designing the next generation of optogenetic tools.
Assuntos
Ânions/metabolismo , Channelrhodopsins/química , Channelrhodopsins/metabolismo , Criptófitas/química , Bacteriorodopsinas/química , Sítios de Ligação , Channelrhodopsins/efeitos da radiação , Cristalografia por Raios X , Condutividade Elétrica , Ativação do Canal Iônico/efeitos da radiação , Transporte de Íons/efeitos da radiação , Modelos Moleculares , Optogenética/métodos , Optogenética/tendências , Retinaldeído/metabolismo , Bases de Schiff/químicaRESUMO
Both designed and natural anion-conducting channelrhodopsins (dACRs and nACRs, respectively) have been widely applied in optogenetics (enabling selective inhibition of target-cell activity during animal behaviour studies), but each class exhibits performance limitations, underscoring trade-offs in channel structure-function relationships. Therefore, molecular and structural insights into dACRs and nACRs will be critical not only for understanding the fundamental mechanisms of these light-gated anion channels, but also to create next-generation optogenetic tools. Here we report crystal structures of the dACR iC++, along with spectroscopic, electrophysiological and computational analyses that provide unexpected insights into pH dependence, substrate recognition, channel gating and ion selectivity of both dACRs and nACRs. These results enabled us to create an anion-conducting channelrhodopsin integrating the key features of large photocurrent and fast kinetics alongside exclusive anion selectivity.
Assuntos
Ânions/metabolismo , Channelrhodopsins/química , Channelrhodopsins/metabolismo , Ativação do Canal Iônico , Optogenética/métodos , Animais , Caenorhabditis elegans , Células Cultivadas , Channelrhodopsins/genética , Channelrhodopsins/efeitos da radiação , Cristalografia por Raios X , Eletrofisiologia , Feminino , Células HEK293 , Hipocampo/citologia , Humanos , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico/efeitos da radiação , Transporte de Íons/efeitos da radiação , Cinética , Masculino , Camundongos , Modelos Moleculares , Neurônios/metabolismo , Especificidade por SubstratoRESUMO
Lysophosphatidic acid (LPA) is a bioactive lipid composed of a phosphate group, a glycerol backbone, and a single acyl chain that varies in length and saturation. LPA activates six class A G-protein-coupled receptors to provoke various cellular reactions. Because LPA signalling has been implicated in cancer and fibrosis, the LPA receptors are regarded as promising drug targets. The six LPA receptors are subdivided into the endothelial differentiation gene (EDG) family (LPA1-LPA3) and the phylogenetically distant non-EDG family (LPA4-LPA6). The structure of LPA1 has enhanced our understanding of the EDG family of LPA receptors. By contrast, the functional and pharmacological characteristics of the non-EDG family of LPA receptors have remained unknown, owing to the lack of structural information. Although the non-EDG LPA receptors share sequence similarity with the P2Y family of nucleotide receptors, the LPA recognition mechanism cannot be deduced from the P2Y1 and P2Y12 structures because of the large differences in the chemical structures of their ligands. Here we determine the 3.2 Å crystal structure of LPA6, the gene deletion of which is responsible for congenital hair loss, to clarify the ligand recognition mechanism of the non-EDG family of LPA receptors. Notably, the ligand-binding pocket of LPA6 is laterally open towards the membrane, and the acyl chain of the lipid used for the crystallization is bound within this pocket, indicating the binding mode of the LPA acyl chain. Docking and mutagenesis analyses also indicated that the conserved positively charged residues within the central cavity recognize the phosphate head group of LPA by inducing an inward shift of transmembrane helices 6 and 7, suggesting that the receptor activation is triggered by this conformational rearrangement.
Assuntos
Lisofosfolipídeos/química , Lisofosfolipídeos/metabolismo , Receptores de Ácidos Lisofosfatídicos/química , Receptores de Ácidos Lisofosfatídicos/metabolismo , Alopecia/congênito , Alopecia/genética , Animais , Sítios de Ligação , Membrana Celular/metabolismo , Cristalografia por Raios X , Células HEK293 , Humanos , Ligantes , Simulação de Acoplamento Molecular , Mutagênese , Filogenia , Estabilidade Proteica , Estrutura Secundária de Proteína , Receptores de Ácidos Lisofosfatídicos/genética , Especificidade por Substrato , Peixe-Zebra/genéticaRESUMO
Krokinobacter eikastus rhodopsin 2 (KR2) is the first light-driven Na(+) pump discovered, and is viewed as a potential next-generation optogenetics tool. Since the positively charged Schiff base proton, located within the ion-conducting pathway of all light-driven ion pumps, was thought to prohibit the transport of a non-proton cation, the discovery of KR2 raised the question of how it achieves Na(+) transport. Here we present crystal structures of KR2 under neutral and acidic conditions, which represent the resting and M-like intermediate states, respectively. Structural and spectroscopic analyses revealed the gating mechanism, whereby the flipping of Asp116 sequesters the Schiff base proton from the conducting pathway to facilitate Na(+) transport. Together with the structure-based engineering of the first light-driven K(+) pumps, electrophysiological assays in mammalian neurons and behavioural assays in a nematode, our studies reveal the molecular basis for light-driven non-proton cation pumps and thus provide a framework that may advance the development of next-generation optogenetics.
Assuntos
Flavobacteriaceae/química , Bombas de Íon/química , Bombas de Íon/efeitos da radiação , Luz , Rodopsina/química , Rodopsina/efeitos da radiação , Sódio/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Concentração de Íons de Hidrogênio , Bombas de Íon/genética , Bombas de Íon/metabolismo , Transporte de Íons/genética , Transporte de Íons/efeitos da radiação , Modelos Biológicos , Modelos Moleculares , Mutagênese/genética , Optogenética , Potássio/metabolismo , Conformação Proteica , Engenharia de Proteínas , Retinaldeído/química , Retinaldeído/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Bases de Schiff , Relação Estrutura-AtividadeRESUMO
Activation of the µ-opioid receptor (µOR) is responsible for the efficacy of the most effective analgesics. To shed light on the structural basis for µOR activation, here we report a 2.1 Å X-ray crystal structure of the murine µOR bound to the morphinan agonist BU72 and a G protein mimetic camelid antibody fragment. The BU72-stabilized changes in the µOR binding pocket are subtle and differ from those observed for agonist-bound structures of the ß2-adrenergic receptor (ß2AR) and the M2 muscarinic receptor. Comparison with active ß2AR reveals a common rearrangement in the packing of three conserved amino acids in the core of the µOR, and molecular dynamics simulations illustrate how the ligand-binding pocket is conformationally linked to this conserved triad. Additionally, an extensive polar network between the ligand-binding pocket and the cytoplasmic domains appears to play a similar role in signal propagation for all three G-protein-coupled receptors.
Assuntos
Receptores Opioides mu/química , Receptores Opioides mu/metabolismo , Regulação Alostérica , Animais , Sítios de Ligação , Cristalografia por Raios X , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Morfinanos/química , Morfinanos/metabolismo , Morfinanos/farmacologia , Estabilidade Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Pirróis/química , Pirróis/metabolismo , Pirróis/farmacologia , Receptor Muscarínico M2/química , Receptores Adrenérgicos beta 2/química , Receptores Opioides mu/agonistas , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/farmacologia , Relação Estrutura-AtividadeRESUMO
Ion-translocating rhodopsins, especially channelrhodopsins (ChRs), have attracted broad attention as a powerful tool to modulate the membrane potential of cells with light (optogenetics). Because of recent biophysical, spectroscopic, and computational studies, including the structural determination of cation and anion ChRs, our understanding of the molecular mechanism underlying light-gated ion conduction has been greatly advanced. In this chapter, I first describe the background of rhodopsin family proteins including ChR, and how the optogenetics technology has been established from the discovery of first ChR in 2002. I later introduce the recent findings of the structure-function relationship of ChR by comparing the crystal structures of cation and anion ChRs. I further discuss the future goal in the fields of ChR research and optogenetic tool development.
Assuntos
Channelrhodopsins/química , Channelrhodopsins/metabolismo , Optogenética , Rodopsina/química , Rodopsina/metabolismo , Channelrhodopsins/genética , Channelrhodopsins/efeitos da radiação , Luz , Potenciais da Membrana , Optogenética/métodos , Rodopsina/genética , Rodopsina/efeitos da radiação , Relação Estrutura-AtividadeRESUMO
Multidrug and toxic compound extrusion (MATE) family transporters are conserved in the three primary domains of life (Archaea, Bacteria and Eukarya), and export xenobiotics using an electrochemical gradient of H(+) or Na(+) across the membrane. MATE transporters confer multidrug resistance to bacterial pathogens and cancer cells, thus causing critical reductions in the therapeutic efficacies of antibiotics and anti-cancer drugs, respectively. Therefore, the development of MATE inhibitors has long been awaited in the field of clinical medicine. Here we present the crystal structures of the H(+)-driven MATE transporter from Pyrococcus furiosus in two distinct apo-form conformations, and in complexes with a derivative of the antibacterial drug norfloxacin and three in vitro selected thioether-macrocyclic peptides, at 2.1-3.0 Å resolutions. The structures, combined with functional analyses, show that the protonation of Asp 41 on the amino (N)-terminal lobe induces the bending of TM1, which in turn collapses the N-lobe cavity, thereby extruding the substrate drug to the extracellular space. Moreover, the macrocyclic peptides bind the central cleft in distinct manners, which correlate with their inhibitory activities. The strongest inhibitory peptide that occupies the N-lobe cavity may pave the way towards the development of efficient inhibitors against MATE transporters.
Assuntos
Antiporters/química , Antiporters/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Pyrococcus furiosus/química , Sequência de Aminoácidos , Apoproteínas/química , Apoproteínas/metabolismo , Ácido Aspártico/química , Cristalografia por Raios X , Análise Mutacional de DNA , Compostos Macrocíclicos/química , Compostos Macrocíclicos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Norfloxacino/química , Norfloxacino/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Conformação Proteica , Prótons , Relação Estrutura-Atividade , Sulfetos/química , Sulfetos/metabolismoRESUMO
Channelrhodopsins (ChRs) are light-gated cation channels derived from algae that have shown experimental utility in optogenetics; for example, neurons expressing ChRs can be optically controlled with high temporal precision within systems as complex as freely moving mammals. Although ChRs have been broadly applied to neuroscience research, little is known about the molecular mechanisms by which these unusual and powerful proteins operate. Here we present the crystal structure of a ChR (a C1C2 chimaera between ChR1 and ChR2 from Chlamydomonas reinhardtii) at 2.3 Å resolution. The structure reveals the essential molecular architecture of ChRs, including the retinal-binding pocket and cation conduction pathway. This integration of structural and electrophysiological analyses provides insight into the molecular basis for the remarkable function of ChRs, and paves the way for the precise and principled design of ChR variants with novel properties.
Assuntos
Cátions/metabolismo , Chlamydomonas reinhardtii/química , Ativação do Canal Iônico/efeitos da radiação , Canais Iônicos/química , Luz , Rodopsina/química , Animais , Bacteriorodopsinas/química , Sítios de Ligação , Bovinos , Chlamydomonas reinhardtii/genética , Cristalografia por Raios X , Canais Iônicos/genética , Canais Iônicos/efeitos da radiação , Modelos Moleculares , Mutação , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/efeitos da radiação , Retinaldeído/metabolismo , Rodopsina/genética , Rodopsina/efeitos da radiação , Bases de Schiff/química , Eletricidade EstáticaRESUMO
Rhodopsins are one of the most studied photoreceptor protein families, and ion-translocating rhodopsins, both pumps and channels, have recently attracted broad attention because of the development of optogenetics. Recently, a new functional class of ion-pumping rhodopsins, an outward Na+ pump, was discovered, and following structural and functional studies enable us to compare three functionally different ion-pumping rhodopsins: outward proton pump, inward Cl- pump, and outward Na+ pump. Here, we review the current knowledge on structure-function relationships in these three light-driven pumps, mainly focusing on Na+ pumps. A structural and functional comparison reveals both unique and conserved features of these ion pumps, and enhances our understanding about how the structurally similar microbial rhodopsins acquired such diverse functions. We also discuss some unresolved questions and future perspectives in research of ion-pumping rhodopsins, including optogenetics application and engineering of novel rhodopsins.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Luz , Rodopsina/metabolismo , Sódio/metabolismo , Animais , Proteínas de Transporte de Cátions/química , Humanos , Modelos Moleculares , Optogenética , Conformação Proteica , Rodopsina/químicaRESUMO
Light-driven outward sodium-pumping rhodopsin (NaR) was recently found in marine bacteria. Krokinobacter eikastus rhodopsin 2 (KR2) actively transports sodium and lithium ions in NaCl and LiCl, respectively, while it pumps protons in KCl. NaR has a conserved NDQ (N112, D116, and Q123 in KR2) motif, and previous studies suggested an important role for N112 in the function of KR2. Here we replaced N112 with 19 different amino acids and studied the molecular properties of the mutants. All mutants exhibited absorption bands from a protonated Schiff base in the λmax range from 508 to 531 nm upon heterologous expression in Escherichia coli, whose ion-pumping activity was measured using pH electrodes. The function of these mutants was classified into three phenotypes: wild-type (WT)-like Na+/H+ compatible pump, exclusive H+ pump, and no pump. Among the 19 mutants, only N112D, -G, -S, and -T showed light-driven Na+ pump activity, N112A, -C, -P, -V, -E, -Q, -I, -L, -M, -F, and -W were exclusively H+ pumps, and N112H, -K, -Y, and -R exhibited no pump activity. The mutants of the no pump function lack a blue-shifted M intermediate, indicating that Schiff base deprotonation is a prerequisite for Na+ and H+ pumps. In contrast, the subsequent red-shifted O intermediate was observed for WT and N112V but absent for N112T and N112A, suggesting that observation of this intermediate depends on kinetics. Although N112D, -G, -S, and -T are able to pump Na+, they also pump H+ in NaCl, where Na+ and H+ pumps compete with each other because of the decreased Na+ uptake efficiency. From these facts, an exclusive Na+ pump in NaCl exists only in WT. We conclude that N112 is one of the functional determinants of NaR.
Assuntos
Asparagina/química , Luz , Rodopsina/química , Sódio/química , Mutação , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Espectrofotometria UltravioletaRESUMO
Channelrhodopsin-2 (ChR2) from the green alga Chlamydomonas reinhardtii functions as a light-gated cation channel that has been developed as an optogenetic tool to stimulate specific nerve cells in animals and control their behavior by illumination. The molecular mechanism of ChR2 has been extensively studied by a variety of spectroscopic methods, including light-induced difference Fourier transform infrared (FTIR) spectroscopy, which is sensitive to structural changes in the protein upon light activation. An atomic structure of channelrhodopsin was recently determined by x-ray crystallography using a chimera of channelrhodopsin-1 (ChR1) and ChR2. Electrophysiological studies have shown that ChR1/ChR2 chimeras are less desensitized upon continuous illumination than native ChR2, implying that there are some structural differences between ChR2 and chimeras. In this study, we applied light-induced difference FTIR spectroscopy to ChR2 and ChR1/ChR2 chimeras to determine the molecular basis underlying these functional differences. Upon continuous illumination, ChR1/ChR2 chimeras exhibited structural changes distinct from those in ChR2. In particular, the protonation state of a glutamate residue, Glu-129 (Glu-90 in ChR2 numbering), in the ChR chimeras is not changed as dramatically as in ChR2. Moreover, using mutants stabilizing particular photointermediates as well as time-resolved measurements, we identified some differences between the major photointermediates of ChR2 and ChR1/ChR2 chimeras. Taken together, our data indicate that the gating and desensitizing processes in ChR1/ChR2 chimeras are different from those in ChR2 and that these differences should be considered in the rational design of new optogenetic tools based on channelrhodopsins.
Assuntos
Chlamydomonas reinhardtii , Luz , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Rodopsina/química , Rodopsina/metabolismo , Sequência de Aminoácidos , Ativação do Canal Iônico , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação Proteica , Estabilidade Proteica , Proteínas Recombinantes de Fusão/genética , Rodopsina/genéticaRESUMO
Proton-dependent oligopeptide transporters (POTs) are major facilitator superfamily (MFS) proteins that mediate the uptake of peptides and peptide-like molecules, using the inwardly directed H(+) gradient across the membrane. The human POT family transporter peptide transporter 1 is present in the brush border membrane of the small intestine and is involved in the uptake of nutrient peptides and drug molecules such as ß-lactam antibiotics. Although previous studies have provided insight into the overall structure of the POT family transporters, the question of how transport is coupled to both peptide and H(+) binding remains unanswered. Here we report the high-resolution crystal structures of a bacterial POT family transporter, including its complex with a dipeptide analog, alafosfalin. These structures revealed the key mechanistic and functional roles for a conserved glutamate residue (Glu310) in the peptide binding site. Integrated structural, biochemical, and computational analyses suggested a mechanism for H(+)-coupled peptide symport in which protonated Glu310 first binds the carboxyl group of the peptide substrate. The deprotonation of Glu310 in the inward open state triggers the release of the bound peptide toward the intracellular space and salt bridge formation between Glu310 and Arg43 to induce the state transition to the occluded conformation.