ABSTRACT
Arrestins recognize different receptor phosphorylation patterns and convert this information to selective arrestin functions to expand the functional diversity of the G protein-coupled receptor (GPCR) superfamilies. However, the principles governing arrestin-phospho-receptor interactions, as well as the contribution of each single phospho-interaction to selective arrestin structural and functional states, are undefined. Here, we determined the crystal structures of arrestin2 in complex with four different phosphopeptides derived from the vasopressin receptor-2 (V2R) C-tail. A comparison of these four crystal structures with previously solved Arrestin2 structures demonstrated that a single phospho-interaction change results in measurable conformational changes at remote sites in the complex. This conformational bias introduced by specific phosphorylation patterns was further inspected by FRET and 1H NMR spectrum analysis facilitated via genetic code expansion. Moreover, an interdependent phospho-binding mechanism of phospho-receptor-arrestin interactions between different phospho-interaction sites was unexpectedly revealed. Taken together, our results provide evidence showing that phospho-interaction changes at different arrestin sites can elicit changes in affinity and structural states at remote sites, which correlate with selective arrestin functions.
Subject(s)
Receptors, Vasopressin/metabolism , beta-Arrestin 1/metabolism , Crystallography, X-Ray , HEK293 Cells , Humans , Mutation , Nuclear Magnetic Resonance, Biomolecular , Phosphopeptides/chemistry , Phosphopeptides/metabolism , Phosphorylation , Protein Conformation, alpha-Helical , Protein Domains/genetics , Receptors, Vasopressin/chemistry , Receptors, Vasopressin/ultrastructure , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , beta-Arrestin 1/genetics , beta-Arrestin 1/isolation & purification , beta-Arrestin 1/ultrastructureABSTRACT
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-ß2 adrenergic receptor/ß-arrestin-1(ß-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 ß-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.
Subject(s)
Arrestin/chemistry , Arrestin/genetics , Arrestin/metabolism , Ligands , Proton Magnetic Resonance Spectroscopy/methods , Binding Sites , Crystallography, X-Ray , Humans , Models, Molecular , Phenylalanine , Protein Binding , Protein Conformation , Receptors, Adrenergic, beta-2/metabolism , Signal Transduction , beta-Arrestin 1/chemistry , beta-Arrestin 1/genetics , beta-Arrestin 1/metabolismABSTRACT
Described is a facile helix-nucleating template based on a tethered aspartic acid at the N-terminus [terminal aspartic acid (TD)]. The nucleating effect of the template is subtly influenced by the substituent at the end of the side-chain-end tether as indicated by circular dichroism, nuclear magnetic resonance, and molecular dynamics simulations. Unlike most nucleating strategies, the N-terminal amine is preserved, thus enabling further modification. Peptidomimetic estrogen receptor modulators (PERMs) constructed using this strategy show improved therapeutic properties. The current strategy can be regarded as a good complement to existing helix-stabilizing methods.
