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

Abstract

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 type B receptor (ET_B), which is implicated in challenging cancers with poor immunotherapy responsiveness. The Streptomyces-derived lasso peptide, RES-701-3, is a selective inhibitor for ET_B 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 ET_B 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 ET_B 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.

Fig. 1. Cryo-EM structure determination of ET_B using a three-point fusion strategy.

a Concept design of the three-point fusion strategy. b Purification of the ET_B-CN-FKBP12 complex. Protein purifications were successfully reproduced at least twice. c, d Representative 2D cryo-EM averages of the ET_B–CN-FKBP12 complex in the apo state (c) and bound to RES-701-3 (d). e, f Cryo-EM density maps and 3D models of the ET_B–CN-FKBP12 complexes in the apo state (e) and complex with RES-701-3 (f), viewed from the side and top.

Fig. 2. Interactions between ET_B and calcineurin.

a Structural comparison of the crystal structure of the calcineurin-FKBP12 complex (PDB 1TCO) and the current apo ET_B-CN-FKBP12 complex. b, c Close-up views of the N-terminus and C-terminus of calcineurin. d Electrostatic surface potentials of ET_B and calcineurin in the apo-ET_B-CN-FKBP12 complex. e Charged residues at the interface of ET_B and calcineurin. f–h Structural comparison of the apo-ET_B-CN-FKBP12 complex and the AF-predicted ET_B-CN structure.

Fig. 3. Structural comparison of inactive ET_B structures.

a–c Superimposition of the apo ET_B-CN-FKBP12 complex and the apo-crystal structure of ET_B-mT4L (PDB 5GLI), viewed from the extracellular side (a) and the membrane plane (b and c). The mT4L and CN-B fusions into ICL3 are shown in (c).

Fig. 4. RES-701-3 binding mode.

a Schematic illustration of the lasso peptide RES-701-3. b Cryo-EM map of RES-701-3. c Residues involved in RES-701-3 binding within 4.5 Å. Black dashed lines indicate hydrogen bonds. d, e Close-up views of W10 (d) and W3 (e). Bulky residues are shown as CPK models.

Fig. 5. Structural comparison of the ET_B receptors.

a–d Structural changes upon binding of ET-1 (a, PDB 8IY5), RES-701-3 (b), bosentan (c, PDB 5 × 93), and IRL2500 (d, PDB 6KIQ), focused on TM6. Apo state and compound-bound structures are colored gray and light-green, respectively. Black arrows indicate conformational changes upon drug binding.

Fig. 6. Binding modes of cyclic peptides.

a Cavity for RES-701-3. The ET_B receptor is shown as a molecular surface. b, c Cavities for MccJ25 in bacterial RNA polymerase (PBD 6N60) (b) and the siderophore receptor FhuA (PBD 4CU4) (c). d Crystal structures of the P. furiosus MATE transporter bound to the thioether-macrocyclic peptides MaD3S (PBD 3VVS) and MaL6 (PBD 3WBN).