Exploring Pairwise Chemical Crosslinking To Study Peptide–Receptor Interactions

Abstract

Pairwise crosslinking is a powerful technique to characterize interactions between G protein coupled receptors and their ligands in the live cell. In this work, the “thiol trapping” method, which exploits the proximity‐enhanced reaction between haloacetamides and cysteine, is examined to identify intermolecular pairs of vicinal positions. By incorporating cysteine into the corticotropin‐releasing factor receptor and either α‐chloro‐ or α‐bromoacetamide groups into its ligands, it is shown that thiol trapping provides highly reproducible signals and a low background, and represents a valid alternative to classical “disulfide trapping”. The method is advantageous if reducing agents are required during sample analysis. Moreover, it can provide partially distinct spatial constraints, thus giving access to a wider dataset for molecular modeling. Finally, by applying recombinant mini‐Gs, GTPγS, and Gαs‐depleted HEK293 cells to modulate Gs coupling, it is shown that yields of crosslinking increase in the presence of elevated levels of Gs.

Structural details of a Class B GPCR-arrestin complex revealed by genetically encoded crosslinkers in living cells

“…Next, we identified intermolecular pairs of proximal PTH1R-arrestin amino acids via pairwise crosslinking. We exploited the reaction between the electrophilic ncAA O -(2-bromoethyl)-tyrosine (BrEtY) and the nucleophilic thiol of canonical cysteine (“thiol trapping” method 40 ), which occurs only when the two groups come into close proximity (Fig. 2a ) 41 .…”

Exploring GPCR‐arrestin interfaces with genetically encoded crosslinkers

“…Position 75/76 is quite intriguing, because Bpa75/76‐βarr1/2 give both a low‐MW band and an activation‐dependent crosslinking band (especially with CRF 1 R, Fig 2). To test whether low‐MW bands are due to intra‐molecular crosslinking, we applied proximity‐induced pair‐wise crosslinking (Fig 5A) (Xiang et al , 2013; Wang, 2017b; Coin, 2018; Seidel et al , 2019). The mildly electrophilic ncAA O‐(2‐bromoethyl)‐tyrosine (BrEtY) (Xiang et al , 2014) was genetically incorporated at position F75 of βarr1.…”

Capture and mass spectrometry analysis of effector-substrate complexes using genetically incorporated photo-crosslinkers in host cells

“…It is also important to notice that failure of crosslinking only means the capture failed, but does not necessarily mean no interaction. 18 The failure may be caused by unsuitable distance, inappropriate orientation, unusual transient interaction, and so on. The side chain lengths of different Uaas are different.…”