Revealing the architecture of protein complexes by an orthogonal approach combining HDXMS, CXMS, and disulfide trapping.

Many cellular functions necessitate structural assemblies of two or more associated proteins. The structural characterization of protein complexes using standard methods, such as X-ray crystallography, is challenging. Herein, we describe an orthogonal approach using hydrogen-deuterium-exchange mass spectrometry (HDXMS), cross-linking mass spectrometry (CXMS), and disulfide trapping to map interactions within protein complexes. HDXMS measures changes in solvent accessibility and hydrogen bonding upon complex formation; a decrease in HDX rate could account for newly formed intermolecular or intramolecular interactions. To distinguish between inter- and intramolecular interactions, we use a CXMS method to determine the position of direct interface regions by trapping intermolecular residues in close proximity to various cross-linkers (e.g., disuccinimidyl adipate (DSA)) of different lengths and reactive groups. Both MS-based experiments are performed on high-resolution mass spectrometers (e.g., an Orbitrap Elite hybrid mass spectrometer). The physiological relevance of the interactions identified through HDXMS and CXMS is investigated by transiently co-expressing cysteine mutant pairs, one mutant on each protein at the discovered interfaces, in an appropriate cell line, such as HEK293. Disulfide-trapped protein complexes are formed within cells spontaneously or are facilitated by addition of oxidation reagents such as H2O2 or diamide. Western blotting analysis, in the presence and absence of reducing reagents, is used to determine whether the disulfide bonds are formed in the proposed complex interface in physiologically relevant milieus. The procedure described here requires 1-2 months. We demonstrate this approach using the ß2-adrenergic receptor-ß-arrestin1 complex as the model system.

Distinct conformations of GPCR-ß-arrestin complexes mediate desensitization, signaling, and endocytosis.

ß-Arrestins (ßarrs) interact with G protein-coupled receptors (GPCRs) to desensitize G protein signaling, to initiate signaling on their own, and to mediate receptor endocytosis. Prior structural studies have revealed two unique conformations of GPCR-ßarr complexes: the "tail" conformation, with ßarr primarily coupled to the phosphorylated GPCR C-terminal tail, and the "core" conformation, where, in addition to the phosphorylated C-terminal tail, ßarr is further engaged with the receptor transmembrane core. However, the relationship of these distinct conformations to the various functions of ßarrs is unknown. Here, we created a mutant form of ßarr lacking the "finger-loop" region, which is unable to form the core conformation but retains the ability to form the tail conformation. We find that the tail conformation preserves the ability to mediate receptor internalization and ßarr signaling but not desensitization of G protein signaling. Thus, the two GPCR-ßarr conformations can carry out distinct functions.

Visualization of arrestin recruitment by a G-protein-coupled receptor.

G-protein-coupled receptors (GPCRs) are critically regulated by ß-arrestins, which not only desensitize G-protein signalling but also initiate a G-protein-independent wave of signalling. A recent surge of structural data on a number of GPCRs, including the ß2 adrenergic receptor (ß2AR)-G-protein complex, has provided novel insights into the structural basis of receptor activation. However, complementary information has been lacking on the recruitment of ß-arrestins to activated GPCRs, primarily owing to challenges in obtaining stable receptor-ß-arrestin complexes for structural studies. Here we devised a strategy for forming and purifying a functional human ß2AR-ß-arrestin-1 complex that allowed us to visualize its architecture by single-particle negative-stain electron microscopy and to characterize the interactions between ß2AR and ß-arrestin 1 using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and chemical crosslinking. Electron microscopy two-dimensional averages and three-dimensional reconstructions reveal bimodal binding of ß-arrestin 1 to the ß2AR, involving two separate sets of interactions, one with the phosphorylated carboxy terminus of the receptor and the other with its seven-transmembrane core. Areas of reduced HDX together with identification of crosslinked residues suggest engagement of the finger loop of ß-arrestin 1 with the seven-transmembrane core of the receptor. In contrast, focal areas of raised HDX levels indicate regions of increased dynamics in both the N and C domains of ß-arrestin 1 when coupled to the ß2AR. A molecular model of the ß2AR-ß-arrestin signalling complex was made by docking activated ß-arrestin 1 and ß2AR crystal structures into the electron microscopy map densities with constraints provided by HDX-MS and crosslinking, allowing us to obtain valuable insights into the overall architecture of a receptor-arrestin complex. The dynamic and structural information presented here provides a framework for better understanding the basis of GPCR regulation by arrestins.