Conformational analysis of β-Arrestin2 pre-activated mutant p44 by hydrogen/ deuterium exchange mass spectrometry

Arrestins have important roles in G protein-coupled receptor (GPCR) signaling including desensitization of GPCRs and G protein-independent signal transduction. Two major intra-molecular interactions, the polar core and the three-element region, maintain arrestins in the basal conformation by connecting the N- and C-domains. Several mutations in these regions convert arrestins to interact with GPCRs in a phosphorylation-independent manner, and p44, the truncation mutation that lacks the C-terminal tail, is one of those pre-activated mutants. Studying the conformation of p44 would provide insight into understanding the structural mechanism of GPCR-arrestin interaction. Previous studies analyzed p44 form of arrestin1 and β-arrestin1 with X-ray crystallography and hydrogen/deuterium exchange mass spectrometry(HDX-MS), and the conformation of R169E, another pre-activated mutant, was analyzed in β-arrestin1 and β-arrestin2 by HDX-MS. Here, we analyzed the conformational dynamics of β-arrestin2 p44 by HDX-MS to have more precise structural information on the pre-activation of arrestins. HDX-MS analysis revealed that p44 has higher deuterium uptake than the basal state in various regions. Together with previous structural studies of pre-activated arrestins, we suggest that β-arrestin1 and β-arrestin2 undergo different conformational changes upon pre-activation and that the effects of each preactivating mutants varies although all of them increased the conformational dynamics compared to the basal state.