Optimizing cryo-EM structural analysis of Gi-coupling receptors via engineered Gt and Nb35 application

Oshima, Hidetaka S.; Sano, Fumiya K.; Akasaka, Hiroaki; Iwama, Aika; Shihoya, Wataru; Nureki, Osamu

doi:10.1016/j.bbrc.2023.149361

“…Next, we inspected the interaction between A 3 R and G i . There are hallmark regions of the interactions between class A G i -coupling receptors, including A 3 R, and G i : ICL2 of a receptor as well as α5 of G i (Akasaka et al, 2022; Hua et al, 2020; Izume et al, 2024; Kato et al, 2019; Okamoto et al, 2021; Oshima et al, 2024; Sano et al, 2023; Yuan et al, 2021; Zhuang et al, 2022). In detail, around α5 of G i , several residues form hydrogen bonds, represented by the interaction between R108 3.50 and C351 H5.23 (superscript indicates the common Gα numbering [CGN] system(Flock et al, 2015)) (Figure 2F).…”

Section: Resultsmentioning

confidence: 99%

“…In detail, around α5 of G i , several residues form hydrogen bonds, represented by the interaction between R108 3.50 and C351 H5.23 (superscript indicates the common Gα numbering [CGN] system(Flock et al, 2015)) (Figure 2F). In the ICL2 of A 3 R, V116 34.51 penetrates into the hydrophobic cavity of G i , which is common in class A GPCRs(Akasaka et al, 2022; Hua et al, 2020; Iwama et al, 2023; Izume et al, 2024; Kato et al, 2019; Nagiri et al, 2021; Okamoto et al, 2021; Oshima et al, 2024; Rasmussen et al, 2011; Sano et al, 2023; Yuan et al, 2021; Zhuang et al, 2022) (Figure 2G). Furthermore, extensive ionic interactions are formed across ICL2.…”

Section: Resultsmentioning

confidence: 99%

Structural insights into the agonist selectivity and structure-based engineering of the adenosine A3 receptor

Oshima,

Ogawa,

Sano

et al. 2024

Preprint

Self Cite

0

1

0

View full text Add to dashboard Cite

Adenosine receptors, expressed across various tissues, play pivotal roles in physiological processes and are implicated in diverse diseases, including neurological disorders and inflammation, highlighting the therapeutic potential of receptor-selective agents. The Adenosine A3 receptor (A3R), the last identified adenosine receptor, is also activated by breakdown products of post-transcriptionally modified tRNA and exhibits dual roles in neuron, heart, and immune cells, and is often overexpressed in tumors, making it a target for anticancer therapy. Despite extensive studies on the other adenosine receptors, the structure and activation mechanism of A3R, especially by selective agonists like N6-methyladenosine (m6A) and namodenoson, remained elusive. Here, we identified N6-isopentenyl adenosine (i6A), a novel A3R-selective ligand, via comprehensive modified adenosine library screening. Cryo-EM analyses of A3R-Gi signaling complexes with two nonselective and three selective agonists revealed the structural basis for A3R activation. We further conducted structure-guided engineering of m6A-insensitive A3R, which would greatly facilitate future discoveries of the physiological functions of the selective activation of A3R by modified adenosines. Our results clarify the selective activation of adenosine receptors, providing the basis for future drug discovery.

show abstract

“…5a). This rotation of W336 6.48 induces and propagates the outward rotation of F332 6.44 within the P 5 .50 I/V 3 .40 F 6.44 motif, ultimately resulting in the intracellular opening 27, 28 . The binding of the antagonist bosentan induces only a minor inward movement in TM6 14 and sterically prevents the rotamer change of W336 6.48 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Structure of a lasso peptide bound ETB receptor provides insights into the mechanism of GPCR inverse agonism

Shihoya,

Akasaka,

Jordan

et al. 2024

Preprint

Self Cite

0

View full text Add to dashboard Cite

Lasso peptides exhibit a unique lariat-like knotted structure imparting exceptional stability and thus show promise as therapeutic agents that target cell-surface receptors. One such receptor is the human endothelin ETB receptor, which is implicated in challenging cancers with poor immunotherapy responsiveness. The Streptomyces-derived lasso peptide, RES-701-3, is a selective inhibitor for ETB and a compelling candidate for therapeutic development. However, meager production from a genetically recalcitrant host has limited further structure-activity relationship studies of this potent inhibitor. Here, we report cryo-electron microscopy structures of ETB receptor in both its apo form and complex with RES-701-3, facilitated by a calcineurin-fusion strategy. Hydrophobic interactions between RES-701-3 and the transmembrane region of the receptor, especially involving two tryptophan residues, play a crucial role in RES-701-3 binding. Furthermore, RES-701-3 prevents conformational changes associated with G-protein coupling, explaining its inverse agonist activity. A comparative analysis with other lasso peptides and their target proteins highlights the potential of lasso peptides as precise drug candidates for G-protein-coupled receptors. This structural insight into RES-701-3 binding to ETB receptor offers valuable information for the development of novel therapeutics targeting this receptor and provides a broader understanding of lasso peptide interactions with human cell-surface receptors.

show abstract

Structure of a lasso peptide bound ETB receptor provides insights into the mechanism of GPCR inverse agonism

Nureki,

Shihoya,

Akasaka

et al. 2024

Preprint

Self Cite

0

View full text Add to dashboard Cite

Lasso peptides exhibit a unique lariat-like knotted structure imparting exceptional stability and thus show promise as therapeutic agents that target cell-surface receptors. One such receptor is the human endothelin ETB receptor, which is implicated in challenging cancers with poor immunotherapy responsiveness. The Streptomyces-derived lasso peptide, RES-701-3, is a selective inhibitor for ETB and a compelling candidate for therapeutic development. However, meager production from a genetically recalcitrant host has limited further structure-activity relationship studies of this potent inhibitor. Here, we report cryo-electron microscopy structures of ETB receptor in both its apo form and complex with RES-701-3, facilitated by a calcineurin-fusion strategy. Hydrophobic interactions between RES-701-3 and the transmembrane region of the receptor, especially involving two tryptophan residues, play a crucial role in RES-701-3 binding. Furthermore, RES-701-3 prevents conformational changes associated with G-protein coupling, explaining its inverse agonist activity. A comparative analysis with other lasso peptides and their target proteins highlights the potential of lasso peptides as precise drug candidates for G-protein-coupled receptors. This structural insight into RES-701-3 binding to ETB receptor offers valuable information for the development of novel therapeutics targeting this receptor and provides a broader understanding of lasso peptide interactions with human cell-surface receptors.

show abstract

Optimizing cryo-EM structural analysis of Gi-coupling receptors via engineered Gt and Nb35 application

Cited by 3 publications

References 34 publications

Structural insights into the agonist selectivity and structure-based engineering of the adenosine A3 receptor

Structural insights into the agonist selectivity and structure-based engineering of the adenosine A3 receptor

Structure of a lasso peptide bound ETB receptor provides insights into the mechanism of GPCR inverse agonism

Structure of a lasso peptide bound ETB receptor provides insights into the mechanism of GPCR inverse agonism

Contact Info

Product

Resources

About