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 β2-adrenergic receptor-β-arrestin1 complex as the model system.

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Figure 1: Schematic view of the orthogonal approach combining HDXMS, CXMS, and disulfide trapping.
Figure 2: Workflow for HDXMS, CXMS, and disulfide-trapping experiments.
Figure 3: Different types of cross-linkers with their reactive groups and lengths of spacer arms.
Figure 4: Fragmentation tuning in HDXMS experiments.
Figure 5: Sample preparation for time-dependent on-exchange HDXMS experiments.
Figure 6: Mapping the interaction interface between β2AR and β-arrestin 1 by HDXMS.
Figure 7: Mapping the interaction interface between β2AR and β-arrestin 1 by CXMS.
Figure 8: Mapping the interaction interface between β2AR and β-arrestin 1 by disulfide trapping.
Figure 9: Revealing the architecture of the β2-adrenergic receptor (β2AR)-βarrestin 1 complex via an orthogonal approach combining HDXMS, CXMS, and disulfide trapping.

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We thank R.J. Lefkowitz (Duke University) and B.K. Kobilka (Stanford University) for invaluable guidance and enthusiastic support, and A.K. Shukla and A.W. Kahsai for stimulating ideas. This work was supported, in part, by US National Institutes of Health grant HL-075443 Proteomics Core support to K.X. This publication was also made possible by seed funding support to K.X. from the Department of Pharmacology and Chemical Biology, the University of Pittsburgh, the Vascular Medicine Institute, the Hemophilia Center of Western Pennsylvania, and the Institute for Transfusion Medicine.

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K.X. conceived the orthogonal approach combining HDXMS, CXMS, and disulfide trapping, and designed the experiments; S.L. conducted the HDXMS experiments; J.Q. conducted the CXMS experiments; M.C. conducted the disulfide-trapping experiments; K.X., Y.Z., H.L., A.B., T.J.C., X.L., Y.X., L.J.C., and S.L. analyzed data and wrote the paper; all authors read, edited, and discussed the paper.

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Correspondence to Kunhong Xiao or Sheng Li.

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Xiao, K., Zhao, Y., Choi, M. et al. Revealing the architecture of protein complexes by an orthogonal approach combining HDXMS, CXMS, and disulfide trapping. Nat Protoc 13, 1403–1428 (2018).

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