Conformational Changes in BID, a Pro-apoptotic BCL-2 Family Member, upon Membrane Binding
2004; Elsevier BV; Volume: 280; Issue: 1 Linguagem: Inglês
10.1074/jbc.m405428200
ISSN1083-351X
AutoresKyoung Joon Oh, Scott Barbuto, Natalie Meyer, Ryung-Suk Kim, R. John Collier, Stanley J. Korsmeyer,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoThe BCL-2 family proteins constitute a critical control point in apoptosis. BCL-2 family proteins display structural homology to channel-forming bacterial toxins, such as colicins, transmembrane domain of diphtheria toxin, and the N-terminal domain of δ-endotoxin. By analogy, it has been hypothesized the BCL-2 family proteins would unfold and insert into the lipid bilayer upon membrane association. We applied the site-directed spin labeling method of electron paramagnetic resonance spectroscopy to the pro-apoptotic member BID. Here we show that helices 6-8 maintain an α-helical conformation in membranes with a lipid composition resembling mitochondrial outer membrane contact sites. However, unlike colicins and the transmembrane domain of diphtheria toxin, these helices of BID are bound to the lipid bilayer without adopting a transmembrane orientation. Our study presents a more detailed model for the reorganization of the structure of tBID on membranes. The BCL-2 family proteins constitute a critical control point in apoptosis. BCL-2 family proteins display structural homology to channel-forming bacterial toxins, such as colicins, transmembrane domain of diphtheria toxin, and the N-terminal domain of δ-endotoxin. By analogy, it has been hypothesized the BCL-2 family proteins would unfold and insert into the lipid bilayer upon membrane association. We applied the site-directed spin labeling method of electron paramagnetic resonance spectroscopy to the pro-apoptotic member BID. Here we show that helices 6-8 maintain an α-helical conformation in membranes with a lipid composition resembling mitochondrial outer membrane contact sites. However, unlike colicins and the transmembrane domain of diphtheria toxin, these helices of BID are bound to the lipid bilayer without adopting a transmembrane orientation. Our study presents a more detailed model for the reorganization of the structure of tBID on membranes. Programmed cell death or apoptosis is a normal physiological form of cell death essential for successful embryonic development and the maintenance of cellular homeostasis (1Danial N.N. Korsmeyer S.J. Cell. 2004; 116: 205-219Abstract Full Text Full Text PDF PubMed Scopus (4060) Google Scholar, 2Opferman J.T. Korsmeyer S.J. Nat. Immun. 2003; 4: 410-415Crossref Scopus (415) Google Scholar, 3Cory S. Huang D.C. Adams J.M. Oncogene. 2003; 22: 8590-8607Crossref PubMed Scopus (1305) Google Scholar). The BCL-2 family is comprised of pro- as well as anti-apoptotic proteins and constitutes critical points in the apoptosis pathway. BID is a pro-apoptotic member of the "BH3-only" subset of the BCL-2 family proteins (4Wang K. Yin X.M. Chao D.T. Milliman C.L. Korsmeyer S.J. Genes Dev. 1996; 10: 2859-2869Crossref PubMed Scopus (808) Google Scholar) that interconnects extrinsic pathway TNFR1 and Fas death signals to the mitochondrial amplification of the intrinsic pathway. Engagement of TNFR1 and Fas activates caspase-8 that cleaves p22 BID within an unstructured loop generating an N-terminal 7-kDa fragment and a C-terminal 15-kDa fragment (5Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3085) Google Scholar, 6Hochman A. Sternin H. Gorodin S. Korsmeyer S. Ziv I. Melamed E. Offen D. J. Neurochem. 1998; 71: 741-748Crossref PubMed Scopus (125) Google Scholar, 7Gross A. Yin X.M. Wang K. Wei M.C. Jockel J. Milliman C. Erdjument-Bromage H. Tempst P. Korsmeyer S.J. J. Biol. Chem. 1999; 274: 1156-1163Abstract Full Text Full Text PDF PubMed Scopus (932) Google Scholar). Cleaved BID then undergoes post-translational modification by myristoylation at a newly generated N-terminal glycine of the p15-kDa fragment (p15 BID or tBID) (8Zha J. Weiler S. Oh K.J. Wei M.C. Korsmeyer S.J. Science. 2000; 290: 1761-1765Crossref PubMed Scopus (477) Google Scholar). Myristoylation of the BID complex p7/myr-p15 serves as a molecular switch facilitating the targeting of BID to the mitochondrion. tBID localizes to mitochondrial contact sites where cardiolipin has been reported to exist (9Lutter M. Fang M. Luo X. Nishijima M. Xie X. Wang X. Nat. Cell Biol. 2000; 2: 754-761Crossref PubMed Scopus (411) Google Scholar, 10Lutter M. Perkins G.A. Wang X. BMC Cell Biol. 2001; 2: 22-30Crossref PubMed Scopus (126) Google Scholar, 11Kim T.H. Zhao Y. Ding W.X. Shin J.N. He X. Seo Y.W. Chen J. Rabinowich H. Amoscato A.A. Yin X.M. Mol. Biol. Cell. 2004; 15: 3061-3072Crossref PubMed Scopus (150) Google Scholar). Targeted p15 BID triggers the homo-oligomerization of multidomain pro-apoptotic BAX or BAK in the mitochondrial outer membrane (MOM) 1The abbreviations used are: MOM, mitochondrial outer membrane; SDSL, site-directed spin labeling; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]; PI, phosphatidylinositol; MCL, monolysocardiolipin; PC tempo, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphotempocholine; n-doxyl-PC, 1-palmitoyl-2-stearoyl(n-doxyl)-sn-glycero-3-phosphocholine where n = 5, 7, or 10; OMCT, mitochondrial outer membrane contact site; OM, mitochondrial outer membrane; DTT, dithiothreitol; OG, n-octyl-β-d-glucopyranoside; MTSL, (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate spin label; NiEDDA, nickel(II)ethylenediamine diacetate; FITC, fluorescein isothiocyanate; GST, glutathione S-transferase; wt, wild type; Mops, 4-morpholinepropane-sulfonic acid; LUVs, large unilamellar vesicles. 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These results indicate that BID, a Bcl-2 family protein, interacts with the lipid bilayer in a mechanism distinct from membrane-translocating bacterial toxins despite their structural similarity. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG), beef liver phosphatidylinositol (PI), beef heart cardiolipin, beef heart monolysocardiolipin (MCL), cholesterol, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphotempocholine (PC tempo), 1-palmitoyl-2-stearoyl(5-doxyl)-sn-glycero-3-phosphocholine (5-doxyl-PC), 1-palmitoyl-2-stearoyl(7-doxyl)-sn-glycero-3-phosphocholine (7-doxyl-PC), and 1-palmitoyl-2-stearoyl(10-doxyl)-sn-glycero-3-phosphocholine (10-doxyl-PC) were purchased from Avanti Polar Lipids. N-Tempoylpalmitamide was synthesized as described (53Shin Y.K. Hubbell W.L. Biophys. J. 1992; 61: 1443-1453Abstract Full Text PDF PubMed Scopus (49) Google Scholar). Mixtures of lipids were prepared in chloroform, divided in 50-mg aliquots, and dried as thin films in glass test tubes under nitrogen gas. These were further dried under vacuum for 16 h and resuspended in buffer A (20 mm Hepes, 150 mm KCl (pH 7.0)). The lipid suspensions were freeze-thawed three times and extruded 15 times through two sheets of polycarbonate membrane with a pore size of 100 nm (Avestin) using an extruder (Avanti Polar Lipids), resulting in large unilamellar vesicles (LUVs) (54Szoka F. Olson F. Heath T. Vail W. Mayhew E. Papahadjopoulos D. Biochim. Biophys. Acta. 1980; 601: 559-571Crossref PubMed Scopus (449) Google Scholar). Vesicles resembling the lipid composition of the mitochondrial outer membrane contact sites, designated as OMCT vesicles, were made with a mixture of POPC, POPE, PI, cholesterol, and cardiolipin in the weight ratio of 41:22:9:8:20 (9Lutter M. Fang M. Luo X. Nishijima M. Xie X. Wang X. Nat. Cell Biol. 2000; 2: 754-761Crossref PubMed Scopus (411) Google Scholar, 52Ardail D. Privat J.P. Egret-Charlier M. Levrat C. Lerme F. Louisot P. J. Biol. Chem. 1990; 265: 18797-18802Abstract Full Text PDF PubMed Google Scholar). Vesicles resembling the mitochondrial outer membrane, designated as OM vesicles, were made without cardiolipin by mixing POPC, POPE, PI, and cholesterol in the weight ratio of 61:22:9:8. Vesicles with 20% POPG or 15% MCL and 5% cardiolipin, designated as OMPG (or OMCL) vesicles, were also made using the same ratio of lipids as in the OMCT except of cardiolipin. For the immersion depth measurements, OMCT vesicles containing trace amounts (1:1000 by weight) of PC tempo, N-tempoylpalmitamide, 5-, 7-, or 10-doxyl-PC were also prepared in buffer A. The phosphate contents of the vesicles were determined as described (55Böttcher C.J.F. van Gent C.M. Pries C. Anal. Chim. Acta. 1961; 24: 203-204Crossref Scopus (854) Google Scholar). Full-length mouse Bid gene was cloned into a modified pET22b vector, which allows the expression of p22 BID as a fusion protein with an N-terminal hexahistidine tag (56Oh K.J. Senzel L. Collier R.J. Finkelstein A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8467-8470Crossref PubMed Scopus (121) Google Scholar). The complete sequence of the N-terminal tag is "MGSSHHHHHHSSGLVPRGSH," in single letter code. The two native cysteine residues Cys-30 and Cys-126 were mutated to serines using QuickChange site-directed mutagenesis kit (Stratagene). In this cysteine-less clone, designated as p22BID30S126S, cysteine mutations were introduced. The nucleotide sequences were verified by sequencing the entire gene. The p22 BID proteins were expressed and purified according to the pET system manual (Novagen). Proteins were purified from the periplasmic extracts by Ni2+ affinity and anion-exchange chromatography using an AKTA fast performance liquid chromatography system (Amersham Biosciences). Proteins were stored at -80 °C in 20 mm Tris buffer (pH 7.5) containing 16% (v/v) glycerol, 10 mm DTT, and ∼200 mm NaCl. The p22 BID proteins with a C-terminal histidine tag (p22BID-Chis) were also produced using the pET22b expression system. The complete sequence of the C-terminal tag is "LEHHHHHH." A cysteine-less clone, p22BID30S126S-Chis, and its derivative with a Gly to Glu substitution mutation at residue 94, p22BID30S126S94E-Chis, were prepared similarly by following the above procedures. A glutathione S-transferase fusion protein of BCL-XL (residues 1-212) lacking the C-terminal transmembrane domain, designated as GST-BCL-XLΔC, was expressed in Escherichia coli BL21 using pGEX2T (Amersham Biosciences) and purified by affinity chromatography with glutathione-agarose beads from Sigma. Caspase-8 was purified as described (57Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1650) Google Scholar). Full-length human BAX protein was purified as described (30Suzuki M. Youle R.J. Tjandra N. Cell. 2000; 103: 645-654Abstract Full Text Full Text PDF PubMed Scopus (909) Google Scholar). Protein concentrations were determined by Bradford assay with Coomassie Brilliant Blue G-250 dye (Bio-Rad) using bovine serum albumin as a standard (58Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217548) Google Scholar). Peptides corresponding to the BH3 domains of wild type and mutant human BID and human BAD were synthesized at the Tufts Peptide Synthesis Core facility. They were purified by reverse-phase high pressure liquid chromatography, and masses were confirmed by mass spectrometry. The amino acid sequences for BH3 peptides for the wild type BID, the mutant BID, and BAD are as follows: EDIIRNIARHLAQVGDSMDR; EDIIRNIARHAAQVGASMDR, and NLWAAQRYGRELRRMSDEFVDSFKK, respectively (the underlines indicate substitution mutations in the sequence). The peptides are modified with acetyl and amino groups at the N and C terminus, respectively. The biological activity of BID proteins was assessed by the cytochrome c release assay (5Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3085) Google Scholar). First, recombinant p22 BID proteins were cleaved with caspase-8. Caspase-8 was mixed with p22 BID (1-2 mg/ml) at 1:100 weight ratio in 20 mm Tris buffer (pH 7.5) containing ∼200 mm NaCl and 10 mm DTT. The reaction mixture was incubated for 16 h at room temperature, resulting in p7/p15 BID complex. Cleavage was verified by PAGE. All the protein samples were diluted to a final concentration of 17.6 μg/ml. Next, mouse liver mitochondria were isolated at 4 °C according to the procedures in the literature with minor modifications (5Luo X. Budihardjo I. Zou H. Slaughter C. Wang X. Cell. 1998; 94: 481-490Abstract Full Text Full Text PDF PubMed Scopus (3085) Google Scholar, 59Ellerby H.M. Martin S.J. Ellerby L.M. Naiem S.S. Rabizadeh S. Salvesen G.S. Casiano C.A. Cashman N.R. Green D.R. Bredesen D.E. J. Neurosci. 1997; 17: 6165-6178Crossref PubMed Google Scholar). Briefly, the liver was removed from a mouse after sacrifice, minced with scissors, and homogenized in a glass homogenizer in 20 ml of ice-cold mitochondria isolation buffer (buffer B) containing 250 mm sucrose, 0.5 mm EGTA, and 10 mm Tris-HCl. Intact cells and nuclei were pelleted by centrifugation at 600 × g for 10 min. The supernatants were further centrifuged at 7,000 × g for 10 min to pellet the mitochondria. The mitochondrial pellets were gently resuspended in 10 ml of buffer B and centrifuged at 7,000 × g for 10 min. The mitochondria were then resuspended gently in 0.3 ml of buffer B. The protein concentration of the mitochondrial preparation was determined after solubilizing the mitochondria in 0.1% deoxycholic acid in phosphate-buffered saline solution. Mitochondria were then diluted to a final concentration of 0.5 mg of mitochondrial protein per ml in buffer C (10 mm Tris-Mops (pH 7.4), 1 mm KH2PO4, 10 μm Tris-EGTA, 5 mm glutamate, 2 mm malate, and 125 mm KCl). Finally, 2 μl of p7/p15 BID was added to 100 μl of the diluted mitochondria, resulting in the BID/mitochondrial protein ratio of 32 pmol/mg. The reaction mixture was incubated at room temperature for 40 min. The reaction mixture was then centrifuged at 12,000 × g for 5 min at 4 °C to pellet the mitochondria. The supernatants were quickly removed, and the pellets were resuspended in the same volume of phosphate-buffered saline containing 0.5% Triton X-100. The amount of cytochrome c in the supernatants or in the pellet fractions was determined in duplicate by an enzyme-linked immunosorbent assay (Quantikine M kit, R&D Systems, Minneapolis, MN). Cysteine mutant proteins of p22 BID (1-2 mg) were reacted with 10-20-fold excess of (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate spin label (MTSL) to result in R1 side chain (Fig. 1C) immediately after removing DTT by gel filtration chromatography in buffer A (20 mm Hepes, 150 mm KCl (pH 7.0)). Unreacted spin label was removed by gel filtration chromatography in buffer A. Proteins were concentrated with centrifugal concentrators with the molecular weight cut-off of 5 kDa (Millipore). In order to spin label p15 BID, p22 BID single-cysteine mutants (∼5 mg) were first cleaved by incubation with caspase-8 in the presence of 10 mm DTT as described above. DTT was then removed by passing the reaction mixture through a PD-10 desalting column (Amersham Biosciences) after pre-equilibrating it with buffer A. MTSL was added at 10-fold excess to the eluted protein solution and reacted for 40-60 min. Unreacted spin label was removed by passing the reaction mixture through a second PD-10 column pre-equilibrated with buffer A. The spin-labeled p7/p15 BID was then loaded onto a column containing 0.5 ml of Ni2+-nitrilotriacetic acid-agarose beads (Qiagen) and washed with 10 ml of buffer A. Spin-labeled p15 BID was eluted with 40 ml of buffer A containing 2% n-octyl-β-d-glucopyranoside (OG, Calbiochem). The eluted spin-labeled p15 BID solution was then concentrated by using a centrifugal concentrator (molecular weight cut-off of 5 kDa, Millipore) to a final volume of ∼2.5 ml. Protein concentration was determined by using a bicinchoninic acid protein assay kit (BCA, Pierce) using bovine serum albumin as a standard (60Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18713) Google Scholar). Large unilamellar vesicles prepared as described above were then added to the concentrated tBID solution at the lipid to protein ratio of 1:10 (w/w). The protein/lipid mixture was placed in a dialysis cassette with a 10-kDa molecular weight cut-off (Pierce) and dialyzed for 16-24 h at 4 °C against 1 liter of buffer A four times to remove the detergent. Protein aggregates that did not incorporate into the membranes were removed by a sucrose step gradient. Briefly, sucrose stock solutions of 2.5, 5.0, 7.5, 10.0, 12.5, and 15% (w/v) were prepared in buffer A containing 0.05% NaN3. A volume of 1 ml of the sucrose stock solutions was successively layered in an ultracentrifuge tube, and the reconstituted p15 BID vesicles were placed on top. This was subjected to centrifugation at 210,000 × g for 7-16 h at 4 °C. Fractions containing reconstituted p15 BID were collected from 5 to 7.5% sucrose bands. These fractions were shown by electron microscopy to contain large and round vesicles, whereas fractions in 10-12.5% sucrose layers contained protein/lipid aggregates. Collected fractions were further centrifuged to pellet the vesicles at 150,000 × g for ∼5 h after diluting the sample in buffer A in a 5-ml ultracentrifuge tube. For immersion depth measurements, p15 BID prepared with BID30S126S was reconstituted in OMCT vesicles containing a trace amount of spin-labeled fatty acid or lipids as described above. After reconstitution, the phosphate content of the vesicles and the protein concentrations were determined as described (55Böttcher C.J.F. van Gent C.M. Pries C. Anal. Chim. Acta. 1961; 24: 203-204Crossref Scopus (854) Google Scholar). EPR spectra were obtained on a Bruker EMX spectrometer using a Bruker High Sensitivity resonator at room temperature. All spectra were recorded at 2-milliwatt incident microwave power using a field modulation of 1.0-1.5 G at 100 kHz. For power saturation experiments, NiEDDA was synthesized as described (45Oh K.J. Altenbach C. Collier R.J. Hubbell W.L. Methods Mol. Biol. 2000; 145: 147-169PubMed Google Scholar, 61Altenbach C. Greenhalgh D.A. Khorana H.G. Hubbell W.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1667-1671Crossref PubMed Scopus (407) Google Scholar). In order to measure the accessibility parameters, Π, of O2 and NiEDDA, power saturation experiments were carried out with a loop-gap resonator (JAGMAR, Krakow, Poland) (45Oh K.J. Altenbach C. Collier R.J. Hubbell W.L. Methods Mol. Biol. 2000; 145: 147-169PubMed Google Scholar, 63Hubbell W.L. Altenbach C. Holst I. Membrane Protein Structure: Experimental Approaches. Oxford University Press, Oxford1994: 244-248Google Scholar, 64Hubbell W.L. Altenbach C. Curr. Opin. Struct. Biol. 1994; 4: 566-573Crossref Scopus (364) Google Scholar, 65Farahbakhsh Z.T. Altenbach C. Hubbell W.L. Photochem. Photobiol. 1992; 56: 1019-1033Crossref PubMed Scopus (124) Google Scholar, 66Hubbell W.L. Froncisz W. Hyde J.S. Rev. Sci. Instrum. 1987; 58: 1879-1886Crossref Scopus (135) Google Scholar). The source of oxygen gas was air-supplied in-house, and the concentration of NiEDDA was 5 mm. N2 gas was used to purge O2 when necessary. In order to measure the immersion depths of membrane-inserted R1 residues, air O2 and 50 mm NiEDDA were used as collision reagents. The range of the incident microwave power was 0.2 to 80 milliwatts for power saturation experiments. Power saturation data were analyzed using the R program (version 1.5.1) (67Ihaka R. Gentleman R. J. Comput. Graph. Stat. 1996; 3: 299-314Google Scholar). Depth calibration curve was determined using the p15 BID-bound vesicles containing spin-labeled lipids (61Altenbach C. Greenhalgh D.A. Khorana H.G. Hubbell W.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1667-1671Crossref PubMed Scopus (407) Google Scholar, 65Farahbakhsh Z.T. Altenbach C. Hubbell W.L. Photochem. Photobiol. 1992; 56: 1019-1033Crossref PubMed Scopus (124) Google Scholar). The depth standards used were PC tempo, N-tempoylpalmitamide, 5-doxyl-PC, 7-doxyl-PC, and 1
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