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

Abstract

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.

Molecular mechanism of the endothelin receptor type B interactions with Gs, Gi, and Gq

“…Owing to the nature of the transmembrane protein, the sequence coverage of the mass spectrometric analysis of ET B was low (41%) (Figure S1C and Figure 3A). Nevertheless, with the analyzed peptic peptides, we observed that the HDX-MS profiles of Gi1 and Gq co-incubated ET B were identical (Supplementary dataset), consistent with the similar high-resolution structures of ET B complexed with Gi1 and Gq (Figure 3D) 12,13 .…”

“…Yet, as described above, the HDX profile showed higher HDX levels at the nucleotide-binding regions in the ETB-coincubated Gαi1 or Gαq (Figure 2A and 2B), supporting ETB-induced GDP release from Gi1 and Gq in the current HDX system. Together, the data suggests the shallow or unstable nature of the ETB-Gi1 or ETB-Gq interaction and explain the necessity for HiBiT tethering strategies for stable ETB-Gi1 or ETB-Gq complex formation 12,13 .…”

“…The high-resolution structures of ET B -Gi1 and ET B -Gq complexes also revealed extensive interaction of the C-terminus of Gα α5 with the receptor (Figure 4A and 4B) 12,13 . However, we did not observe HDX changes at the C-terminus of Gα upon co-incubation with ET B (Figure 2A and 2B, Supplementary dataset).…”

“…Recent cryo-EM studies of ETB in complex with Gq or Gi1 have provided insight into coupling mechanisms and G protein-subtype selectivity between ETB and G proteins [12][13][14] . In these studies, however, owing to weak or unstable association between ETB and G proteins, the ETB-G protein complexes were forced to be made using a HiBiT tethering strategy 15 .…”

Structure and dynamics of the RF-amide QRFP receptor GPR103

“…Finally, we compared the GPR103-Gq complex structure with those of other Class A GPCRs bound to various G-proteins. In terms of the binding angles for α5h, GPR103-Gq naturally exhibits similarity to Gq-coupled GPCRs rather than Gs-and Gi-coupled GPCRs 13,16,17,[22][23][24] (Fig. 2d, e).…”