Use of Volta Phase Plate single particle cryo-electron microscopy for determination of active state G protein-coupled receptor structure

Wootten D, Liang L, Khoshouei M, Glukhova A, Draper Joyce C, Christopoulos A and Sexton PM

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria.

Cryo-electron microscopy (cryo-EM) has gained prominence as a method of choice for determination of structure for difficult to crystallise membrane proteins, including active state, transducer complexed G protein-coupled receptors (GPCRs). Class GPCRs bind critically important physiological peptides of 30-40 amino acids, and are important targets for major diseases including diabetes, obesity and osteoporosis. Our laboratory has recently applied Volta Phase Plate (VPP) single particle cryo-EM to determine structures of class GPCRs in complex with their canonical transducers, heterotrimeric Gs proteins. This methodology has been applied to minimally modified receptors, including the human calcitonin receptor, the glucagon-like peptide-1 receptor (GLP-1R), and the calcitonin-gene-related peptide receptor (CGRPR), which consists of the calcitonin-related receptor and a single pass transmembrane protein, receptor-activity modifying protein 1 (RAMP1). These studies reveal common macromolecular changes associated with class B GPCR activation and G protein coupling, receptor specific-differences in peptide hormone binding and critical structural insights into GPCR modulation by RAMPs. Moreover, for the GLP-1R we have solved structures bound to different biased peptide agonists that reveal conformational variances within the receptor that can be linked to distinct efficacy for Gs signalling. More recently, we have solved the structure of the class A adenosine A1 receptor bound to its endogenous agonist adenosine and a Gi2 heterotrimeric protein, revealing novel insights regarding GPCR G protein selectivity. Collectively, this work highlights the power of VPP cryo-EM for GPCR structure determination and the similarities and diversities in modes of receptor activation between members of related subfamilies.