Reconstitution and Characterization of BCL-2 Family Proteins in Lipid Bilayer Nanodiscs
The BCL-2 family proteins are key regulators of programmed cell death or apoptosis, and represent important targets for the development of anticancer drugs. Because their functions are intimately connected with intracellular membranes, it is important to perform structural and activity studies in precisely characterized samples that include phospholipids and capture the features of the native physiological environment as closely as possible. NMR studies and activity assays based on lipid bilayer nanodiscs are ideally suited for this purpose: they enable the conformations and interactions of these proteins to be probed at atomic resolution in their membrane-associated states. Here we describe detailed protocols for generating the protein components and the reconstituted nanodisc samples suitable for NMR studies and functional assays. The protocols focus on the BCL-2 family protein BCL-XL, a dominant inhibitor of programmed cell death and a major anticancer drug target. The protocols are relatively straightforward. Provided care is taken to ensure protein integrity and sample homogeneity, BCL-XL can be readily reconstituted in nanodiscs, with its hydrophobic C-terminal tail anchored through the nanodisc lipid bilayer, and its folded N-terminal head and ligand binding pocket exposed to the aqueous solution. We anticipate that BCL-2 samples prepared with these protocols will advance structural and mechanistic studies for this important protein family.
Key wordsBCL-2 Apoptosis Membrane Nanodisc Structure NMR
This work was supported by grants from the National Institutes of Health (R01CA179087, R01GM100265, P41EB002031, and P30CA030199).
- 1.Oltersdorf T, Elmore SW, Shoemaker AR, Armstrong RC, Augeri DJ, Belli BA, Bruncko M, Deckwerth TL, Dinges J, Hajduk PJ, Joseph MK, Kitada S, Korsmeyer SJ, Kunzer AR, Letai A, Li C, Mitten MJ, Nettesheim DG, Ng S, Nimmer PM, O'Connor JM, Oleksijew A, Petros AM, Reed JC, Shen W, Tahir SK, Thompson CB, Tomaselli KJ, Wang B, Wendt MD, Zhang H, Fesik SW, Rosenberg SH (2005) An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature 435:677–681CrossRefGoogle Scholar
- 3.Lessene G, Czabotar PE, Sleebs BE, Zobel K, Lowes KN, Adams JM, Baell JB, Colman PM, Deshayes K, Fairbrother WJ, Flygare JA, Gibbons P, Kersten WJ, Kulasegaram S, Moss RM, Parisot JP, Smith BJ, Street IP, Yang H, Huang DC, Watson KG (2013) Structure-guided design of a selective BCL-X(L) inhibitor. Nat Chem Biol 9:390–397CrossRefGoogle Scholar
- 6.Gonzalez-Garcia M, Perez-Ballestero R, Ding L, Duan L, Boise LH, Thompson CB, Nunez G (1994) bcl-XL is the major bcl-x mRNA form expressed during murine development and its product localizes to mitochondria. Development 120:3033–3042Google Scholar
- 7.Fang W, Rivard JJ, Mueller DL, Behrens TW (1994) Cloning and molecular characterization of mouse bcl-x in B and T lymphocytes. J Immunol 153:4388–4398Google Scholar
- 21.Cavanagh J, Fairbrother WJ, Palmer AG, Skelton NJ (1996) Protein NMR spectroscopy: principles and practice. Academic Press, San DiegoGoogle Scholar
- 26.Johnson BA, Blevins RA (1995) NMR view: a computer program for the visualization and analysis of NMR data. J Biomol NMR 5:603–614Google Scholar
- 27.Goddard TD, Kneller DG (2004) SPARKY 3. University of California, San FranciscoGoogle Scholar