Effective Application of Bicelles for Conformational Analysis of G Protein-Coupled Receptors by Hydrogen/Deuterium Exchange Mass Spectrometry

B American Society for Mass Spectrometry, 2015 J. Am. Soc. Mass Spectrom. (2015) 26:808Y817 DOI: 10.1007/s13361-015-1083-4 RESEARCH ARTICLE Effective Application of Bicelles for Conformational Analysis of G Protein-Coupled Receptors by Hydrogen/Deuterium Exchange Mass Spectrometry Nguyen Minh Duc,1 Yang Du,2 Thor S. Thorsen,2 Su Youn Lee,1 Cheng Zhang,2,3 Hideaki Kato,2 Brian K. Kobilka,2 Ka Young Chung1 1 School of Pharmacy, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon, 440740, South Korea Department of Molecular and Cellular Physiology, Stanford University Medical School, 297 Campus Drive, Beckman Center, Stanford, CA 94305, USA 3 Present Address: Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, E 1358 Thomas E. Starzl Biomedical Science Tower, 200 Lothrop St., Pittsburgh, PA 15213, USA 2 Abstract. G protein-coupled receptors (GPCRs) have important roles in physiology and pathology, and 40% of drugs currently on the market target GPCRs for the treatment of various diseases. Because of their therapeutic importance, the structural mechanism of GPCR signaling is of great interest in the field of drug discovery. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a useful tool for analyzing ligand binding sites, the protein–protein interaction interface, and conformational changes of proteins. However, its application to GPCRs has been limited for various reasons, including the hydrophobic nature of GPCRs and the use of detergents in their preparation. In the present study, we tested the application of bicelles as a means of solubilizing GPCRs for HDX-MS studies. GPCRs (e.g., β2-adrenergic 2 receptor [β AR], μ-opioid receptor, and protease-activated receptor 1) solubilized in bicelles produced better sequence coverage (greater than 90%) than GPCRs solubilized in n-dodecyl-β-D-maltopyranoside (DDM), suggesting that bicelles are a more effective method of solubilization for HDX-MS studies. The HDX-MS profile of β2AR in bicelles showed that transmembrane domains (TMs) undergo lower deuterium uptake than intracellular or extracellular regions, which is consistent with the fact that the TMs are highly ordered and embedded in bicelles. The overall HDX-MS profiles of β2AR solubilized in bicelles and in DDM were similar except for intracellular loop 3. Interestingly, we detected EX1 kinetics, an important phenomenon in protein dynamics, at the C-terminus of TM6 in β2AR. In conclusion, we suggest the application of bicelles as a useful method for solubilizing GPCRs for conformational analysis by HDX-MS. Keywords: GPCR, HDX-MS, Bicelles, Detergent, Conformation, EX1 Received: 16 September 2014/Revised: 15 January 2015/Accepted: 16 January 2015/Published Online: 5 March 2015 Introduction G -protein coupled receptors (GPCRs) are the most important class of membrane receptors with over 800 identified to date in humans, many of which are involved in diseases, Nguyen Minh Duc and Yang Du contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s13361-015-1083-4) contains supplementary material, which is available to authorized users. Correspondence to: Brian Kobilka; e-mail: , Ka Chung; e-mail: such as oncologic, psychiatric, metabolic, neurodegenerative, cardiovascular, and infectious diseases [1, 2]. Approximately 40%–50% of drugs circulating in the market target GPCRs for the treatment of various diseases [2]. Ligand binding induces conformational changes of GPCRs, which in turn regulate interactions with downstream signaling molecules, such as heterotrimeric G-proteins or β-arrestins [3, 4]. Understanding the conformational changes that are induced in GPCRs upon activation or inactivation would greatly advance our understanding of the mechanism of activation and inactivation induced by endogenous or synthetic ligands, and might ultimately lead to the design of more effective and less toxic drugs. N. M. Duc et al.: Application of Bicelles for GPCR HDX-MS Hence, enormous efforts have been invested in the characterization of the structure of GPCRs. X-ray crystallography and NMR spectroscopy are standard techniques for obtaining highresolution structures of proteins. Recent breakthroughs in obtaining high-resolution X-ray crystal structures of GPCRs provide the most comprehensive insights into the unique functional properties of these receptors in both inactive and active states [5]. Although X-ray crystallography gives an important three-dimensional overview of structure, it has certain limitations. The X-ray crystal structure represents a single static conformational state, giving little information about conformational changes or dynamics. Another major limitation is the crystallization process for GPCR engineering, which requires a lot of effort and time, and selection of a ligand and detergent [5, 6]. Additionally, the introduction of nonfunctional insertions, truncations, or point mutations into native GPCRs might affect the endogenous conformation. More importantly, the conditions under which proteins function are generally not compatible with the conditions required for X-ray diffraction. NMR has restrictions associated with protein size and sample preparation, such as expression and isotope labeling of proteins [7], and the application of NMR to structural studies of GPCRs is currently very limited. Therefore, other techniques are needed in order to study the conformation of GPCRs. Hydrogen/deuterium exchange mass spectrometry (HDXMS) measures the exchange rates of peptide amide hydrogen with deuterium in the solvent. In folded proteins, the exchange rate varies depending on the conformation of the proteins [8, 9]; exposed or highly dynamic regions show rapid exchange rates whereas excluded and rigid regions show slow exchange rates [8, 9]. Thus, HDX-MS has been successfully used to study conformational changes [10, 11], the protein–protein interaction interface, protein–small molecule interaction sites, and protein folding [12, 13]. Previously, the Griffin group presented methodology for HDX-MS analysis of β2-adrenergic receptor (β2AR) [14] and analyzed ligand-dependent perturbation of the conformational ensemble of β2AR by HDX-MS [15]. Their study showed approximately 71% sequence coverage but the transmembrane (TM) regions were mostly not covered [14, 15]. Other studies analyzed the conformational changes of GPCR-interacting molecules (e.g., G proteins and β-arrestin) upon binding to GPCRs by HDX-MS, but conformational information on the GPCRs themselves is limited [16–18]. The low sequence coverage in the TM regions in the study by the Griffin group and the even lower sequence coverage when analyzed with GPCR-interacting molecules might reflect the technical challenges associated with studying membrane proteins by mass spectrometry. GPCRs are insoluble and unstable membrane proteins, and the use of detergents is obviously required for the (...truncated)