Biology subjects

Sano, F. K.

Publications and source records attributed to Sano, F. K..

7 recordsLinked to original sources

bioRxiv2024

Molecular basis of ligand promiscuity, structural mimicry, and atypical dimerization in the chemokine receptors

Selectivity of natural agonists for their cognate receptors is one of the hallmarks of the members of GPCR family, and it is crucial for the specificity of downstream signal-transduction. However, this selectivity often breaks down in the chemokine receptor subfamily, wherein a high degree of promiscuity is observed with one receptor recognizing multiple chemokines and one chemokine binding to multiple receptors. The molecular determinants of such a striking promiscuity for natural ligands in the chemokine-chemokine receptor system remain mostly elusive and represent an important knowledge gap in our current understanding. Here, we carry out a comprehensive transducer-coupling analysis, testing all known C-X-C chemokines on every C-X-C type chemokine receptor, to generate a global fingerprint of the selectivity and promiscuity encoded within this system. Taking lead from our finding, we determined cryo-EM structures of the most promiscuous receptor, CXCR2, in complex with every interacting chemokine, and deciphered the conserved molecular signatures and distinct binding modalities. While most chemokines position themselves on the receptor as a dimer, CXCL6 exhibits a monomeric binding pose induced by a previously unanticipated reorientation of its carboxyl-terminal -helix, leading to disruption of the dimer interface. Surprisingly, one of the chemokines, CXCL5, induces a ligand-swapped dimer of CXCR2, the first of its kind observed in class A GPCRs, wherein each protomer of the ligand engages its own receptor without any discernible receptor-receptor interface. These unique observations provide a possible structural mechanism for inherent functional specialization encoded in chemokines despite their convergence to a common receptor. Furthermore, we also determined cryo-EM structures of CXCR3 in complex with G-protein-biased and {beta}-arrestin-biased small molecule agonists that elucidate distinct allosteric modulations in the receptor driving their divergent transducer-coupling bias. Guided by structural analysis and experimental validation, we discover that in contrast to previously held notion, small molecule agonists of CXCR3 display robust agonism at CXCR7, an intrinsically biased, {beta}-arrestin-coupled receptor, making them first-in-class dual agonists for chemokine receptors with exclusive {beta}arr-bias at CXCR7. Taken together, our study provides molecular insights into ligand promiscuity and signaling bias at the chemokine receptors, and also demonstrates a proof of principle that naturally encoded structural mimicry can be recapitulated using synthetic pharmacophores with potential implications for developing novel therapeutics.

biochemistry↗

bioRxiv2024

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

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.

biochemistry↗

bioRxiv2023

Structure and dynamics of the RF-amide QRFP receptor GPR103

Pyroglutamylated RF amide peptide (QRFP) is a type of peptide hormone with a C-terminal RF-amide motif. QRFP selectively activates class-A categorized GPCR, GPR103 to exert various physiological functions such as energy metabolism and appetite regulation. Here, we report the cryo-electron microscopy structure of the QRFP-GPR103-Gq complex at 3.3 [A] resolution. Unlike class-A GPCR, QRFP adopts an extended structure baring no secondary structure, with its N-terminal and C-terminal sides recognized by extracellular and transmembrane domains, respectively, of GPR103. The C-terminal heptapeptide of QRFP penetrates into the orthosteric pocket to act in receptor activation. Particularly, the residues that recognize the RF-amide are highly conserved in the RF-amide receptors. Notably, the unique N-terminal helix-loop-helix of the receptor traps the N-terminal side of QRFP with the pendulum-like motion to guide QRFP into the ligand-binding pocket. 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 QRFP. 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. 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.

biochemistry↗

bioRxiv2023

Optimizing Cryo-EM Structural Analysis of Gi-coupling Receptors via Engineered Gt and Nb35 Application

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 eGt, which exhibits binding affinity to modified nanobody Nb35, the cryo-EM structure of Rhodopsin-eGt 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. HighlightsO_LIThe study introduces the engineered G protein subunit eGT, which enhances the resolution of GPCR-G protein structures by suppressing G protein conformational fluctuations and is particularly beneficial for Gi-coupled receptors. C_LIO_LIThe cryo-EM structure of the ETB receptor complexed with eGt-Nb35 reveals improved map quality, reduced anisotropy, and isotropic density distribution, increasing the accuracy of structural analysis. C_LIO_LIStructural comparison between ETB-Gi and ETB-eGt reveals similar receptor-G protein interactions, demonstrating the utility of eGt-Nb35 for studying GPCR-Gi complexes and the potential for broader applications within the Gi family. C_LI

bioengineering↗

bioRxiv2023

Molecular insights into atypical modes of beta-arrestin interaction with seven transmembrane receptors

{beta}-arrestins are multifunctional proteins that are critically involved in regulating spatio-temporal aspects of GPCR signaling. The interaction of {beta}-arrestins with GPCRs is typically conceptualized in terms of receptor activation and phosphorylation primarily in the carboxyl-terminus. Interestingly however, there are several GPCRs that harbor majority of phosphorylation sites in their 3rd intracellular loop (ICL3) instead of carboxyl-terminus but still robustly engage {beta}-arrestins. Moreover, there are several 7TMRs that are now characterized as intrinsically-biased, {beta}-arrestin-coupled receptors (ACRs) due to lack of functional G-protein-coupling but robust {beta}-arrestin binding leading to functional outcomes. The molecular basis of {beta}-arrestin interaction and activation upon binding to these types of 7TMRs is currently elusive, and it represents a major knowledge gap in our current understanding of this signaling system. Here, we present seven cryo-EM structures of {beta}-arrestins in basal state, activated by the muscarinic M2 receptor (M2R) through its ICL3, and a {beta}-arrestin-coupled receptor known as decoy D6 receptor (D6R). These structural snapshots combined with biochemical, cellular, and biophysical experiments including HDX-MS and MD simulation provide novel insights into the ability of {beta}-arrestins to preferentially select specific phosphorylation patterns in the receptors, and also illuminate the structural diversity in 7TMR-{beta}-arrestin interaction. Surprisingly, we also observe that the carboxyl-terminus of {beta}-arrestin2 but not {beta}-arrestin1 undergoes structural transition from a {beta}-strand to -helix upon activation by D6R, which may preclude the core-interaction with the activated receptor. Taken together, our study elucidates previously unappreciated aspects of 7TMR-{beta}-arrestin interaction, and provides important mechanistic clues about how the two isoforms of {beta}-arrestins can recognize and regulate a large repertoire of GPCRs.

biochemistry↗

bioRxiv2023

Structural basis for lysophosphatidylserine recognition by GPR34

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 either the LysoPS analogue S3E-LysoPS, which contains an ethoxy group at the sn-1 position, or M1, a derivative of S3E-LysoPS in which oleic acid is substituted with a metabolically stable aromatic fatty acid surrogate. In both structures, 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, and the aromatic fatty acid surrogate of M1 forms stable hydrophobic interactions with the cavity, thus acting as a superagonist. 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, suggesting its short signal duration. Overall, we anticipate the present structures will pave the way for development of novel anticancer drugs that specifically target GPR34.

biochemistry↗

bioRxiv2023

Cryo-EM structure of the endothelin-1-ETB-Gi complex

The endothelin ETB receptor is a promiscuous G-protein coupled receptor, activated by vasoactive peptide endothelins. ETB signaling induces reactive astrocytes in the brain and vasorelaxation in vascular smooth muscle, and thus ETB agonists are expected to be utilized for neuroprotection and improved anti-tumor drug delivery. Here, we report a cryo-electron microscopy structure of the endothelin-1-ETB-Gi complex at 2.8-[A] 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, which is essential for G-protein activation, is not conserved in ETB, resulting in a unique structural change upon G-protein activation. As Compared with other GPCR-G-protein complexes, ETB binds Gi at the shallowest position, thus expanding the diversity of G-protein binding. This structural information will facilitate the elucidation of G-protein activation and the rational design of ETB-agonists.

biochemistry↗