Structure and dynamics of the active Gs-coupled human secretin receptor

Abstract

The class B secretin GPCR (SecR) has broad physiological effects, with target potential for treatment of metabolic and cardiovascular disease. Molecular understanding of SecR binding and activation is important for its therapeutic exploitation. We combined cryo-electron microscopy, molecular dynamics, and biochemical cross-linking to determine a 2.3 Å structure, and interrogate dynamics, of secretin bound to the SecR:Gs complex. SecR exhibited a unique organization of its extracellular domain (ECD) relative to its 7-transmembrane (TM) core, forming more extended interactions than other family members. Numerous polar interactions formed between secretin and the receptor extracellular loops (ECLs) and TM helices. Cysteine-cross-linking, cryo-electron microscopy multivariate analysis and molecular dynamics simulations revealed that interactions between peptide and receptor were dynamic, and suggested a model for initial peptide engagement where early interactions between the far N-terminus of the peptide and SecR ECL2 likely occur following initial binding of the peptide C-terminus to the ECD.

Structure and dynamics of semaglutide and taspoglutide bound GLP-1R-Gs complexes

“…Similar to most peptide agonists present in active class B1 GPCR complex structures (Zhao et al 2019, Dong et al 2020, Qiao et al 2020, both semaglutide and taspoglutide adopted a continuous helix with their C-terminus bound to the ECD and N-terminus inserted deeply into the TM core (Figures 1 and S4). The N terminus of GLP-1, particularly residues at position 7, 8, 9, 10, 12, 13 and 15, as well as several residues at the C terminus are crucial for GLP-1 binding and/or activity (Adelhorst et al 1994).…”

“…As described above, the static consensus structures for taspoglutide, semaglutide and GLP-1 bound, active GLP-1Rs are remarkably similar. For other class B1 GPCRs, we have demonstrated that the active complexes are dynamic and that dynamics can contribute to the pharmacological behaviour of bound ligands (Dong et al 2020. To understand and visualize the dynamic motions in GLP-1R complexes, 3D variability analysis implemented in cryoSPARC was performed.…”

“…Advances in cryo-electron microscopy (cryo-EM) have enabled structure determination of GPCRs coupled to heterotrimeric G proteins (Garcia-Nafria and Tate 2019), with recently solved cryo-EM structures of class B1 GPCRs achieving global resolutions of 2.5 Å or below (Liang, Belousoff, Fletcher, et al 2020, Dong et al 2020, Zhang et al 2020). Cryo-EM can also capture ensembles of conformations present during vitrification, providing insight into the dynamic behaviour of proteins (Lau et al 2018, Murata and Wolf 2018), a crucial factor in molecular understanding of agonist binding and GPCR activation.…”

Evolving cryo-EM structural approaches for GPCR drug discovery

“…This was confirmed in the 3D variance analysis of the conformational dynamics of the complex comparing principal components of motion for complexes with and without Nb35, with the greatest difference observed for the relative motion between the G and G interface. Our application of 3D variance analysis to recently solved structures of class B GPCRs, including adenomedullin-1 (AM1) and AM2 receptors 19 , the secretin receptor 20 and GLP-1R bound to peptide and small molecule agonists 14 has revealed common rocking and rotational motions of the G protein relative to the receptors. This is consistent with transient formation and breakage of interactions that are likely to be important in receptor-mediated G protein recruitment and activation, and low resolution for parts of ICL3 and the extension of H8 for class B GPCRs 3,8,9,15,[19][20][21][22][23][24][25][26][27][28] are consistent with transient interactions between these domains and the G protein.…”

“…A key conformational change required nucleotide exchange is outward movement of the AHD of Gs, relative to the ras-like domain. The AHD of Gαs in the nucleotide-free state is highly mobile, thereby occupying different positions around the ras-like domain 30 , and it is poorly resolved in most consensus cryo-EM maps of solved class B GPCR-G protein complexes 3,8,9,15,[19][20][21][22][23][24][25][26][27][28] . When bound with Nb35, 3D multivariate analysis of PF 06882961-GLP-1R-DNGs complex revealed translational motion between open ("up" position) and more closed ("down" position) conformations of the AHD.…”