Conformational Dynamics of Activation for the Pentameric Complex of Dimeric G Protein-Coupled Receptor and Heterotrimeric G Protein

Abstract

Summary Photoactivation of rhodopsin (Rho), a G protein-coupled receptor (GPCR), causes conformational changes that provide a specific binding site for the rod G protein, Gt. In this work we employed structural mass spectrometry (MS) techniques to elucidate the structural changes accompanying transition of ground state Rho to photoactivated Rho (Rho*) and in the pentameric complex between dimeric Rho* and heterotrimeric Gt. Observed differences in hydroxyl radical labeling and deuterium uptake between Rho* and the (Rho*)2-Gt complex suggest that photoactivation causes structural relaxation of Rho following its initial tightening upon Gt coupling. In contrast, nucleotide-free Gt in the complex is significantly more accessible to deuterium uptake allowing it to accept GTP and mediating complex dissociation. Thus, we provide direct evidence that in the critical step of signal amplification, Rho* and Gt exhibit dissimilar conformational changes when they are coupled in the (Rho*)2-Gt complex.

Less is More: Membrane Protein Digestion Beyond Urea–Trypsin Solution for Next-level Proteomics

“…These peptides were each reproduced by several hundred independent digestions of apo and various ligand-bound ␤ 2 AR from multiple batches of purifications (Ref (6) and unpublished data). The DDM solution also successfully reproduced high coverage for other GPCRs in other laboratories (76). However, devised for direct HDX, DLT did not address extensive PTMs and Cys, as they did not pose a problem in the ␤ 2 AR crystallizable construct: All PTM-bearing domains were either genetically truncated (intracellular C-terminal tail and loop 3) or deglycosylated (two adjacent extracellulardomain N-glycosylations) in purification (6).…”

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