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1.
Mol Cell ; 2024 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-39232582

RESUMEN

Channelrhodopsins are microbial light-gated ion channels that can control the firing of neurons in response to light. Among several cation channelrhodopsins identified in Guillardia theta (GtCCRs), GtCCR4 has higher light sensitivity than typical channelrhodopsins. Furthermore, GtCCR4 shows superior properties as an optogenetic tool, such as minimal desensitization. Our structural analyses of GtCCR2 and GtCCR4 revealed that GtCCR4 has an outwardly bent transmembrane helix, resembling the conformation of activated G-protein-coupled receptors. Spectroscopic and electrophysiological comparisons suggested that this helix bend in GtCCR4 omits channel recovery time and contributes to high light sensitivity. An electrophysiological comparison of GtCCR4 and the well-characterized optogenetic tool ChRmine demonstrated that GtCCR4 has superior current continuity and action-potential spike generation with less invasiveness in neurons. We also identified highly active mutants of GtCCR4. These results shed light on the diverse structures and dynamics of microbial rhodopsins and demonstrate the strong optogenetic potential of GtCCR4.

2.
Nat Commun ; 15(1): 5569, 2024 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-38956111

RESUMEN

Vitamin C plays important roles as a cofactor in many enzymatic reactions and as an antioxidant against oxidative stress. As some mammals including humans cannot synthesize vitamin C de novo from glucose, its uptake from dietary sources is essential, and is mediated by the sodium-dependent vitamin C transporter 1 (SVCT1). Despite its physiological significance in maintaining vitamin C homeostasis, the structural basis of the substrate transport mechanism remained unclear. Here, we report the cryo-EM structures of human SVCT1 in different states at 2.5-3.5 Å resolutions. The binding manner of vitamin C together with two sodium ions reveals the counter ion-dependent substrate recognition mechanism. Furthermore, comparisons of the inward-open and occluded structures support a transport mechanism combining elevator and distinct rotational motions. Our results demonstrate the molecular mechanism of vitamin C transport with its underlying conformational cycle, potentially leading to future industrial and medical applications.


Asunto(s)
Ácido Ascórbico , Microscopía por Crioelectrón , Transportadores de Sodio Acoplados a la Vitamina C , Humanos , Transportadores de Sodio Acoplados a la Vitamina C/metabolismo , Transportadores de Sodio Acoplados a la Vitamina C/química , Transportadores de Sodio Acoplados a la Vitamina C/genética , Ácido Ascórbico/metabolismo , Ácido Ascórbico/química , Transporte Biológico , Sodio/metabolismo , Modelos Moleculares , Multimerización de Proteína , Unión Proteica , Células HEK293 , Conformación Proteica
3.
Nat Commun ; 15(1): 4769, 2024 Jun 19.
Artículo en Inglés | MEDLINE | ID: mdl-38897996

RESUMEN

Pyroglutamylated RF-amide peptide (QRFP) is a peptide hormone with a C-terminal RF-amide motif. QRFP selectively activates a class A G-protein-coupled receptor (GPCR) GPR103 to exert various physiological functions such as energy metabolism and appetite regulation. Here, we report the cryo-electron microscopy structure of the QRFP26-GPR103-Gq complex at 3.19 Å resolution. QRFP26 adopts an extended structure bearing no secondary structure, with its N-terminal and C-terminal sides recognized by extracellular and transmembrane domains of GPR103 respectively. This movement, reminiscent of class B1 GPCRs except for orientation and structure of the ligand, is critical for the high-affinity binding and receptor specificity of QRFP26. Mutagenesis experiments validate the functional importance of the binding mode of QRFP26 by GPR103. Structural comparisons with closely related receptors, including RY-amide peptide-recognizing GPCRs, revealed conserved and diversified peptide recognition mechanisms, providing profound insights into the biological significance of RF-amide peptides. Collectively, this study not only advances our understanding of GPCR-ligand interactions, but also paves the way for the development of novel therapeutics targeting metabolic and appetite disorders and emergency medical care.


Asunto(s)
Microscopía por Crioelectrón , Receptores Acoplados a Proteínas G , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/química , Humanos , Células HEK293 , Unión Proteica , Ligandos , Péptidos y Proteínas de Señalización Intercelular
4.
J Biochem ; 176(1): 1-10, 2024 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-38498911

RESUMEN

G-protein-coupled receptors (GPCRs) constitute a prominent superfamily in humans and are categorized into six classes (A-F) that play indispensable roles in cellular communication and therapeutics. Nonetheless, their structural comprehension has been limited by challenges in high-resolution data acquisition. This review highlights the transformative impact of cryogenic electron microscopy (cryo-EM) on the structural determinations of GPCR-G-protein complexes. Specific technologies, such as nanobodies and mini-G-proteins, stabilize complexes and facilitate structural determination. We discuss the structural alterations upon receptor activation in different GPCR classes, revealing their diverse mechanisms. This review highlights the robust foundation for comprehending GPCR function and pave the way for future breakthroughs in drug discovery and therapeutic targeting.


Asunto(s)
Microscopía por Crioelectrón , Receptores Acoplados a Proteínas G , Microscopía por Crioelectrón/métodos , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/química , Conformación Proteica , Animales , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/metabolismo , Anticuerpos de Dominio Único/inmunología , Modelos Moleculares
5.
Nat Commun ; 15(1): 902, 2024 Feb 07.
Artículo en Inglés | MEDLINE | ID: mdl-38326347

RESUMEN

GPR34 is a recently identified G-protein coupled receptor, which has an immunomodulatory role and recognizes lysophosphatidylserine (LysoPS) as a putative ligand. Here, we report cryo-electron microscopy structures of human GPR34-Gi complex bound with one of two ligands bound: either the LysoPS analogue S3E-LysoPS, or M1, a derivative of S3E-LysoPS in which oleic acid is substituted with a metabolically stable aromatic fatty acid surrogate. The ligand-binding pocket is laterally open toward the membrane, allowing lateral entry of lipidic agonists into the cavity. The amine and carboxylate groups of the serine moiety are recognized by the charged residue cluster. The acyl chain of S3E-LysoPS is bent and fits into the L-shaped hydrophobic pocket in TM4-5 gap, and the aromatic fatty acid surrogate of M1 fits more appropriately. Molecular dynamics simulations further account for the LysoPS-regioselectivity of GPR34. Thus, using a series of structural and physiological experiments, we provide evidence that chemically unstable 2-acyl LysoPS is the physiological ligand for GPR34. Overall, we anticipate the present structures will pave the way for development of novel anticancer drugs that specifically target GPR34.


Asunto(s)
Ácidos Grasos , Lisofosfolípidos , Humanos , Microscopía por Crioelectrón , Ácidos Grasos/metabolismo , Ligandos , Lisofosfolípidos/metabolismo , Receptores Lisofosfolípidos/agonistas , Receptores Lisofosfolípidos/metabolismo
6.
Science ; 383(6678): 101-108, 2024 01 05.
Artículo en Inglés | MEDLINE | ID: mdl-38175886

RESUMEN

ß-arrestins (ßarrs) are multifunctional proteins involved in signaling and regulation of seven transmembrane receptors (7TMRs), and their interaction is driven primarily by agonist-induced receptor activation and phosphorylation. Here, we present seven cryo-electron microscopy structures of ßarrs either in the basal state, activated by the muscarinic receptor subtype 2 (M2R) through its third intracellular loop, or activated by the ßarr-biased decoy D6 receptor (D6R). Combined with biochemical, cellular, and biophysical experiments, these structural snapshots allow the visualization of atypical engagement of ßarrs with 7TMRs and also reveal a structural transition in the carboxyl terminus of ßarr2 from a ß strand to an α helix upon activation by D6R. Our study provides previously unanticipated molecular insights into the structural and functional diversity encoded in 7TMR-ßarr complexes with direct implications for exploring novel therapeutic avenues.


Asunto(s)
Dominios y Motivos de Interacción de Proteínas , Receptores Acoplados a Proteínas G , beta-Arrestinas , beta-Arrestinas/química , Microscopía por Crioelectrón , Receptores Acoplados a Proteínas G/química , Transducción de Señal , Conformación Proteica en Lámina beta , Conformación Proteica en Hélice alfa , Humanos
7.
Biochem Biophys Res Commun ; 693: 149361, 2024 01 22.
Artículo en Inglés | MEDLINE | ID: mdl-38128244

RESUMEN

Cryo-EM single particle analysis has recently facilitated the high-resolution structural determination of numerous GPCR-G complexes. Diverse methodologies have been devised with this trend, and in the case of GPCR-Gi complexes, scFv16, an antibody that recognizes the intricate interface of the complex, has been mainly implemented to stabilize the complex. However, owing to their flexibility and heterogeneity, structural determinations of GPCR-Gi complexes remain both challenging and resource-intensive. By employing eGαt, which exhibits binding affinity to modified nanobody Nb35, the cryo-EM structure of Rhodopsin-eGαt complex was previously reported. Using this modified G protein, we determined the structure of the ETB-eGt complex bound to the modified Nb35. The determined structure of ETB receptor was the same as the previously reported ETB-Gi complex, and the resulting dataset demonstrated significantly improved anisotropy. This modified G protein will be utilized for the structural determination of other GPCR-Gi complexes.


Asunto(s)
Proteínas de Unión al GTP , Rodopsina , Microscopía por Crioelectrón , Proteínas de Unión al GTP/metabolismo , Rodopsina/metabolismo , Anticuerpos/metabolismo
8.
J Biochem ; 174(4): 317-325, 2023 Sep 29.
Artículo en Inglés | MEDLINE | ID: mdl-37491722

RESUMEN

Endothelins and their receptors, type A (ETA) and type B (ETB), modulate vital cellular processes, including growth, survival, invasion and angiogenesis, through multiple G proteins. This review highlights the structural determinations of these receptors by X-ray crystallography and cryo-electron microscopy, and their activation mechanisms by endothelins. Explorations of the conformational changes upon receptor activation have provided insights into the unique G-protein coupling feature of the endothelin receptors. The review further delves into the binding modes of the clinical antagonist and the inverse agonists. These findings significantly contribute to understanding the mechanism of G-protein activation and have potential implications for drug development, particularly in the context of vasodilatory antagonists and agonists targeting the endothelin receptors.


Asunto(s)
Agonismo Inverso de Drogas , Endotelinas , Microscopía por Crioelectrón , Endotelinas/metabolismo , Receptores de Endotelina/química , Receptores de Endotelina/metabolismo , Transducción de Señal
9.
Elife ; 122023 04 25.
Artículo en Inglés | MEDLINE | ID: mdl-37096326

RESUMEN

The endothelin ETB receptor is a promiscuous G-protein coupled receptor that is activated by vasoactive peptide endothelins. ETB signaling induces reactive astrocytes in the brain and vasorelaxation in vascular smooth muscle. Consequently, ETB agonists are expected to be drugs for neuroprotection and improved anti-tumor drug delivery. Here, we report the cryo-electron microscopy structure of the endothelin-1-ETB-Gi complex at 2.8 Å resolution, with complex assembly stabilized by a newly established method. Comparisons with the inactive ETB receptor structures revealed how endothelin-1 activates the ETB receptor. The NPxxY motif, essential for G-protein activation, is not conserved in ETB, resulting in a unique structural change upon G-protein activation. Compared with other GPCR-G-protein complexes, ETB binds Gi in the shallowest position, further expanding the diversity of G-protein binding modes. This structural information will facilitate the elucidation of G-protein activation and the rational design of ETB agonists.


Asunto(s)
Endotelina-1 , Endotelinas , Endotelina-1/metabolismo , Microscopía por Crioelectrón , Receptor de Endotelina B/metabolismo , Endotelinas/metabolismo , Proteínas de Unión al GTP/metabolismo
10.
Nature ; 616(7956): 390-397, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-37020030

RESUMEN

The class 2 type V CRISPR effector Cas12 is thought to have evolved from the IS200/IS605 superfamily of transposon-associated TnpB proteins1. Recent studies have identified TnpB proteins as miniature RNA-guided DNA endonucleases2,3. TnpB associates with a single, long RNA (ωRNA) and cleaves double-stranded DNA targets complementary to the ωRNA guide. However, the RNA-guided DNA cleavage mechanism of TnpB and its evolutionary relationship with Cas12 enzymes remain unknown. Here we report the cryo-electron microscopy (cryo-EM) structure of Deinococcus radiodurans ISDra2 TnpB in complex with its cognate ωRNA and target DNA. In the structure, the ωRNA adopts an unexpected architecture and forms a pseudoknot, which is conserved among all guide RNAs of Cas12 enzymes. Furthermore, the structure, along with our functional analysis, reveals how the compact TnpB recognizes the ωRNA and cleaves target DNA complementary to the guide. A structural comparison of TnpB with Cas12 enzymes suggests that CRISPR-Cas12 effectors acquired an ability to recognize the protospacer-adjacent motif-distal end of the guide RNA-target DNA heteroduplex, by either asymmetric dimer formation or diverse REC2 insertions, enabling engagement in CRISPR-Cas adaptive immunity. Collectively, our findings provide mechanistic insights into TnpB function and advance our understanding of the evolution from transposon-encoded TnpB proteins to CRISPR-Cas12 effectors.


Asunto(s)
Proteínas Bacterianas , Microscopía por Crioelectrón , Elementos Transponibles de ADN , Deinococcus , Endodesoxirribonucleasas , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/ultraestructura , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , ADN/química , ADN/genética , ADN/metabolismo , ADN/ultraestructura , Elementos Transponibles de ADN/genética , ARN Guía de Sistemas CRISPR-Cas/química , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo , ARN Guía de Sistemas CRISPR-Cas/ultraestructura , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/ultraestructura , Deinococcus/enzimología , Deinococcus/genética , Especificidad por Sustrato
11.
Nature ; 615(7952): 535-540, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36859551

RESUMEN

Energy transfer from light-harvesting ketocarotenoids to the light-driven proton pump xanthorhodopsins has been previously demonstrated in two unique cases: an extreme halophilic bacterium1 and a terrestrial cyanobacterium2. Attempts to find carotenoids that bind and transfer energy to abundant rhodopsin proton pumps3 from marine photoheterotrophs have thus far failed4-6. Here we detected light energy transfer from the widespread hydroxylated carotenoids zeaxanthin and lutein to the retinal moiety of xanthorhodopsins and proteorhodopsins using functional metagenomics combined with chromophore extraction from the environment. The light-harvesting carotenoids transfer up to 42% of the harvested energy in the violet- or blue-light range to the green-light absorbing retinal chromophore. Our data suggest that these antennas may have a substantial effect on rhodopsin phototrophy in the world's lakes, seas and oceans. However, the functional implications of our findings are yet to be discovered.


Asunto(s)
Organismos Acuáticos , Procesos Fototróficos , Bombas de Protones , Rodopsinas Microbianas , Organismos Acuáticos/metabolismo , Organismos Acuáticos/efectos de la radiación , Bacterias/metabolismo , Bacterias/efectos de la radiación , Carotenoides/metabolismo , Color , Cianobacterias/metabolismo , Cianobacterias/efectos de la radiación , Procesos Heterotróficos/efectos de la radiación , Luz , Océanos y Mares , Procesos Fototróficos/efectos de la radiación , Bombas de Protones/metabolismo , Bombas de Protones/efectos de la radiación , Rodopsinas Microbianas/metabolismo , Rodopsinas Microbianas/efectos de la radiación , Zeaxantinas/metabolismo , Zeaxantinas/efectos de la radiación , Luteína/metabolismo , Luteína/efectos de la radiación , Metagenoma , Lagos
12.
Nat Commun ; 13(1): 5417, 2022 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-36109516

RESUMEN

Lysophosphatidic acid receptor 1 (LPA1) is one of the six G protein-coupled receptors activated by the bioactive lipid, lysophosphatidic acid (LPA). LPA1 is a drug target for various diseases, including cancer, inflammation, and neuropathic pain. Notably, LPA1 agonists have potential therapeutic value for obesity and urinary incontinence. Here, we report a cryo-electron microscopy structure of the active human LPA1-Gi complex bound to ONO-0740556, an LPA analog with more potent activity against LPA1. Our structure elucidated the details of the agonist binding mode and receptor activation mechanism mediated by rearrangements of transmembrane segment 7 and the central hydrophobic core. A structural comparison of LPA1 and other phylogenetically-related lipid-sensing GPCRs identified the structural determinants for lipid preference of LPA1. Moreover, we characterized the structural polymorphisms at the receptor-G-protein interface, which potentially reflect the G-protein dissociation process. Our study provides insights into the detailed mechanism of LPA1 binding to agonists and paves the way toward the design of drug-like agonists targeting LPA1.


Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Neuralgia , Receptores del Ácido Lisofosfatídico , Microscopía por Crioelectrón , Humanos , Receptores del Ácido Lisofosfatídico/metabolismo
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