DeSiphering receptor core-induced and ligand-dependent conformational changes in arrestin via genetic encoded trimethylsilyl 1H-NMR probe

Abstract

Characterization of the dynamic conformational changes in membrane protein signaling complexes by nuclear magnetic resonance (NMR) spectroscopy remains challenging. Here we report the site-specific incorporation of 4-trimethylsilyl phenylalanine (TMSiPhe) into proteins, through genetic code expansion. Crystallographic analysis revealed structural changes that reshaped the TMSiPhe-specific amino-acyl tRNA synthetase active site to selectively accommodate the trimethylsilyl (TMSi) group. The unique up-field 1H-NMR chemical shift and the highly efficient incorporation of TMSiPhe enabled the characterization of multiple conformational states of a phospho-β2 adrenergic receptor/β-arrestin-1(β-arr1) membrane protein signaling complex, using only 5 μM protein and 20 min of spectrum accumulation time. We further showed that extracellular ligands induced conformational changes located in the polar core or ERK interaction site of β-arr1 via direct receptor transmembrane core interactions. These observations provided direct delineation and key mechanism insights that multiple receptor ligands were able to induce distinct functionally relevant conformational changes of arrestin.

The role of structural dynamics in GPCR‐mediated signaling

“…In addition to the GPCR-G protein complexes, there is emerging evidence that GPCR-b-arrestin complexes show ligand-dependent structural heterogeneity that may lead to distinct downstream effects [156][157][158]. In the available cryo-EM structures of the NTS1R, M 2 R, and b 1 AR in complex with b-arrestin 1, the receptors couple to b-arrestin 1 via two distinct interfaces [132][133][134].…”

“…The effect of ligands on the core interaction between GPCRs and arrestins has been recently investigated by site‐specific incorporation of unnatural amino acids into β‐arrestin 1 in combination with 1‐dimensional 1 H‐NMR measurements [158]. Binding of arrestin to the β 2 AR in the presence of ligands with different efficacy but identical C‐terminal phosphorylation patterns on the receptor revealed ligand‐dependent conformational changes in the polar core of arrestin, a conserved network of charged residues that is important for stabilization of the inactive state of arrestin.…”

“…Moreover, multiple ligand‐dependent conformational states were detected by NMR at one arrestin site that has been implicated in scaffolding the extracellular signaling‐regulated kinase (ERK) [181]. Although the conformational states did not correlate with the ligand efficacy for G protein activation or arrestin‐mediated receptor internalization, the results suggest that the orthosteric ligand on the receptor can allosterically modulate the conformations of the engaged arrestin with distinct functional outputs [158].…”

“…Taken together, these computational and biophysical studies indicate that orthosteric ligands of GPCRs can modulate the binding and activation of arrestins [158,159,165,167,174]. They do so by impacting the GRK‐mediated phosphorylation of the C terminus of GPCRs or by stabilizing distinct receptor conformations on the intracellular side of the transmembrane bundle.…”

Structural studies of phosphorylation-dependent interactions between the V2R receptor and arrestin-2

“…The recruitment of arrestin by GPCRs is normally preceded by the phosphorylation of GPCRs by a group of GPCR kinases (GRKs), which generates diverse phosphorylation patterns that determine distinct arrestin functions 1 , 4 – 7 , 9 , 10 . Several GRK members, such as GRK2 and GRK3, are known to be activated by Gβγ subunits and thus bridge the G protein activation and arrestin pathways 11 – 13 .…”