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1.
Proc Natl Acad Sci U S A ; 121(44): e2405659121, 2024 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-39441630

RESUMO

Cys-loop receptors are a large superfamily of pentameric ligand-gated ion channels with various physiological roles, especially in neurotransmission in the central nervous system. Among them, zinc-activated channel (ZAC) is a Zn2+-activated ion channel that is widely expressed in the human body and is conserved among eukaryotes. Due to its gating by extracellular Zn2+, ZAC has been considered a Zn2+ sensor, but it has undergone minimal structural and functional characterization since its molecular cloning. Among the families in the Cys-loop receptor superfamily, only the structure of ZAC has yet to be determined. Here, we determined the cryo-EM structure of ZAC in the apo state and performed structure-based mutation analyses. We identified a few residues in the extracellular domain whose mutations had a mild impact on Zn2+ sensitivity. The constriction site in the ion-conducting pore differs from the one in other Cys-loop receptor structures, and further mutational analysis identified a key residue that is important for ion selectivity. In summary, our work provides a structural framework for understanding the ion-conducting mechanism of ZAC.


Assuntos
Microscopia Crioeletrônica , Receptores de Canais Iônicos de Abertura Ativada por Ligante com Alça de Cisteína , Zinco , Zinco/metabolismo , Humanos , Receptores de Canais Iônicos de Abertura Ativada por Ligante com Alça de Cisteína/metabolismo , Receptores de Canais Iônicos de Abertura Ativada por Ligante com Alça de Cisteína/química , Receptores de Canais Iônicos de Abertura Ativada por Ligante com Alça de Cisteína/genética , Modelos Moleculares , Mutação , Conformação Proteica , Ativação do Canal Iônico
2.
PLoS Biol ; 22(4): e3002601, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38656967

RESUMO

Uptake of thiosulfate ions as an inorganic sulfur source from the environment is important for bacterial sulfur assimilation. Recently, a selective thiosulfate uptake pathway involving a membrane protein YeeE (TsuA) in Escherichia coli was characterized. YeeE-like proteins are conserved in some bacteria, archaea, and eukaryotes. However, the precise function of YeeE, along with its potential partner protein in the thiosulfate ion uptake pathway, remained unclear. Here, we assessed selective thiosulfate transport via Spirochaeta thermophila YeeE in vitro and characterized E. coli YeeD (TsuB) as an adjacent and essential protein for YeeE-mediated thiosulfate uptake in vivo. We further showed that S. thermophila YeeD possesses thiosulfate decomposition activity and that a conserved cysteine in YeeD was modified to several forms in the presence of thiosulfate. Finally, the crystal structures of S. thermophila YeeE-YeeD fusion proteins at 3.34-Å and 2.60-Å resolutions revealed their interactions. The association was evaluated by a binding assay using purified S. thermophila YeeE and YeeD. Based on these results, a model of the sophisticated uptake of thiosulfate ions by YeeE and YeeD is proposed.


Assuntos
Escherichia coli , Sulfurtransferases , Tiossulfatos , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico/genética , Cristalografia por Raios X , Cisteína/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ligação Proteica , Sulfurtransferases/genética , Sulfurtransferases/metabolismo , Tiossulfatos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo
3.
Elife ; 122024 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-38578670

RESUMO

P2X receptors are extracellular ATP-gated ion channels that form homo- or heterotrimers and consist of seven subtypes. They are expressed in various tissues, including neuronal and nonneuronal cells, and play critical roles in physiological processes such as neurotransmission, inflammation, pain, and cancer. As a result, P2X receptors have attracted considerable interest as drug targets, and various competitive inhibitors have been developed. However, although several P2X receptor structures from different subtypes have been reported, the limited structural information of P2X receptors in complex with competitive antagonists hampers the understanding of orthosteric inhibition, hindering the further design and optimization of those antagonists for drug discovery. We determined the cryogenic electron microscopy (cryo-EM) structures of the mammalian P2X7 receptor in complex with two classical competitive antagonists of pyridoxal-5'-phosphate derivatives, pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonate) (PPNDS) and pyridoxal phosphate-6-azophenyl-2',5'-disulfonic acid (PPADS), and performed structure-based mutational analysis by patch-clamp recording as well as molecular dynamics (MD) simulations. Our structures revealed the orthosteric site for PPADS/PPNDS, and structural comparison with the previously reported apo- and ATP-bound structures showed how PPADS/PPNDS binding inhibits the conformational changes associated with channel activation. In addition, structure-based mutational analysis identified key residues involved in the PPNDS sensitivity of P2X1 and P2X3, which are known to have higher affinity for PPADS/PPNDS than other P2X subtypes.


Assuntos
Trifosfato de Adenosina , Simulação de Dinâmica Molecular , Animais , Trifosfato de Adenosina/química , Mamíferos
4.
Nat Commun ; 14(1): 6437, 2023 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-37833294

RESUMO

P2X receptors are ATP-activated cation channels, and the P2X4 subtype plays important roles in the immune system and the central nervous system, particularly in neuropathic pain. Therefore, P2X4 receptors are of increasing interest as potential drug targets. Here, we report the cryo-EM structures of the zebrafish P2X4 receptor in complex with two P2X4 subtype-specific antagonists, BX430 and BAY-1797. Both antagonists bind to the same allosteric site located at the subunit interface at the top of the extracellular domain. Structure-based mutational analysis by electrophysiology identified the important residues for the allosteric inhibition of both zebrafish and human P2X4 receptors. Structural comparison revealed the ligand-dependent structural rearrangement of the binding pocket to stabilize the binding of allosteric modulators, which in turn would prevent the structural changes of the extracellular domain associated with channel activation. Furthermore, comparison with the previously reported P2X structures of other subtypes provided mechanistic insights into subtype-specific allosteric inhibition.


Assuntos
Receptores Purinérgicos P2X4 , Peixe-Zebra , Animais , Humanos , Peixe-Zebra/metabolismo , Receptores Purinérgicos P2X4/genética , Receptores Purinérgicos P2X4/metabolismo , Sítio Alostérico , Trifosfato de Adenosina/metabolismo
5.
EMBO Rep ; 22(9): e52911, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34338432

RESUMO

Cilia are thin microtubule-based protrusions of eukaryotic cells. The swimming of ciliated protists and sperm cells is propelled by the beating of cilia. Cilia propagate the flow of mucus in the trachea and protect the human body from viral infections. The main force generators of ciliary beating are the outer dynein arms (ODAs) which attach to the doublet microtubules. The bending of cilia is driven by the ODAs' conformational changes caused by ATP hydrolysis. Here, we report the native ODA complex structure attaching to the doublet microtubule by cryo-electron microscopy. The structure reveals how the ODA complex is attached to the doublet microtubule via the docking complex in its native state. Combined with coarse-grained molecular dynamic simulations, we present a model of how the attachment of the ODA to the doublet microtubule induces remodeling and activation of the ODA complex.


Assuntos
Dineínas do Axonema , Dineínas , Dineínas do Axonema/metabolismo , Axonema/metabolismo , Cílios/metabolismo , Microscopia Crioeletrônica , Dineínas/metabolismo , Humanos , Microtúbulos/metabolismo
6.
Sci Adv ; 6(35): eaba7637, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32923628

RESUMO

We have demonstrated that a bacterial membrane protein, YeeE, mediates thiosulfate uptake. Thiosulfate is used for cysteine synthesis in bacteria as an inorganic sulfur source in the global biological sulfur cycle. The crystal structure of YeeE at 2.5-Å resolution reveals an unprecedented hourglass-like architecture with thiosulfate in the positively charged outer concave side. YeeE is composed of loops and 13 helices including 9 transmembrane α helices, most of which show an intramolecular pseudo 222 symmetry. Four characteristic loops are buried toward the center of YeeE and form its central region surrounded by the nine helices. Additional electron density maps and successive molecular dynamics simulations imply that thiosulfate can remain temporally at several positions in the proposed pathway. We propose a plausible mechanism of thiosulfate uptake via three important conserved cysteine residues of the loops along the pathway.

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