Investigating GPCR-ligand interaction by chemical crosslinking and mass spectrometry

Authors

• BX Huang
• J-W Lee
• H-Y Kim

Abstract

The orphan G protein-coupled receptor 110 (GPR110) belongs to the adhesion GPCR class that is distinctly characterized by the long N-terminus. We have recently identified a lipid ligand that interacts with GPR110. In an effort to understand the molecular basis of this GPCR-ligand interaction, we probe the conformational change of GPR110 using chemical crosslinking and mass spectrometry. Immunopurified HA-GPR110 (human) from HEK cells was treated with or without the ligand followed by incubation with disuccinimidyl suberate (DSS, a lysine-specific crosslinker). The samples were subjected to SDS-PAGE, tryptic digestion, nanoLC-ESI-MS/MS, and label-free quantitation. The MS-based approach identified thirteen through-space crosslinked lysine pairs in the N-terminal domain of the receptor, including K29-K38, K151-K73, K151-K254, K151-K442, K240-K187, K240-K254, K442-K240, K442-K254, K442-K398, K442-K427, K398-K427, K398-K438, and K427-K438. A through-space crosslinking between the intracellular loop 3 (K783) and the C-terminal (K852), along with two long-span loop-links (K864-K873 and K852-K860) within the C-terminal region, was also identified. The data indicated that the α-carbon distance of each of these crosslinked lysine pairs is within ~24 Å, the maximum crosslinking length of the crosslinker. Upon interacting with the lipid ligand, the crosslinking of K427-K438, K442-K427 and K398-K438 increased significantly, suggesting that the local structure of the N-terminal segment involving K427, K438, K398 and K442 becomes more folded upon the ligand binding. Our results represent the first experimental data for the three-dimensional structure of GPR110 at different stages. The ligand-induced change in the crosslinking profile provides valuable information for computer modeling or molecular dynamics simulations of the GPCR-ligand interaction.

Scientific Focus Area: Structural Biology

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