Endothelins and their receptors, ETA and 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.
Cryo-EM advances in GPCR structure determination Shihoya, Wataru; Iwama, Aika; Sano, Fumiya K ...
Journal of Biochemistry/The journal of biochemistry,
07/2024, Letnik:
176, Številka:
1
Journal Article
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Abstract 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.
The class 2 type V CRISPR effector Cas12 is thought to have evolved from the IS200/IS605 superfamily of transposon-associated TnpB proteins
. Recent studies have identified TnpB proteins as miniature ...RNA-guided DNA endonucleases
. 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.
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.
Abstract 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.
Abstract 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-G q 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.
Abstract
Lysophosphatidic acid receptor 1 (LPA
1
) is one of the six G protein-coupled receptors activated by the bioactive lipid, lysophosphatidic acid (LPA). LPA
1
is a drug target for various ...diseases, including cancer, inflammation, and neuropathic pain. Notably, LPA
1
agonists have potential therapeutic value for obesity and urinary incontinence. Here, we report a cryo-electron microscopy structure of the active human LPA
1
-G
i
complex bound to ONO-0740556, an LPA analog with more potent activity against LPA
1
. 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 LPA
1
and other phylogenetically-related lipid-sensing GPCRs identified the structural determinants for lipid preference of LPA
1
. 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 LPA
1
binding to agonists and paves the way toward the design of drug-like agonists targeting LPA
1
.
The endothelin ET
B
receptor is a promiscuous G-protein coupled receptor that is activated by vasoactive peptide endothelins. ET
B
signaling induces reactive astrocytes in the brain and ...vasorelaxation in vascular smooth muscle. Consequently, ET
B
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-ET
B
-G
i
complex at 2.8 Å resolution, with complex assembly stabilized by a newly established method. Comparisons with the inactive ET
B
receptor structures revealed how endothelin-1 activates the ET
B
receptor. The NPxxY motif, essential for G-protein activation, is not conserved in ET
B
, resulting in a unique structural change upon G-protein activation. Compared with other GPCR-G-protein complexes, ET
B
binds G
i
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 ET
B
agonists.
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-G
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.
Energy transfer from light-harvesting ketocarotenoids to the light-driven proton pump xanthorhodopsins has been previously demonstrated in two unique cases: an extreme halophilic bacterium
and a ...terrestrial cyanobacterium
. Attempts to find carotenoids that bind and transfer energy to abundant rhodopsin proton pumps
from marine photoheterotrophs have thus far failed
. 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.
