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• W3150454900 abstract "•BAK monomers are activated by C12E8 detergent to form activated dimers•The crystal structure of these activated BAK dimers is described•The structure provides direct evidence for BAK conformational changes on activation•The active dimer has an extended hydrophobic surface that disrupts lipid membranes A body of data supports the existence of core (α2–α5) dimers of BAK and BAX in the oligomeric, membrane-perturbing conformation of these essential apoptotic effector molecules. Molecular structures for these dimers have only been captured for truncated constructs encompassing the core domain alone. Here, we report a crystal structure of BAK α2–α8 dimers (i.e., minus its flexible N-terminal helix and membrane-anchoring C-terminal segment) that has been obtained through the activation of monomeric BAK with the detergent C12E8. Core dimers are evident, linked through the crystal by contacts via latch (α6–α8) domains. This crystal structure shows activated BAK dimers with the extended latch domain present. Our data provide direct evidence for the conformational change converting BAK from inert monomer to the functional dimer that destroys mitochondrial integrity. This dimer is the smallest functional unit for recombinant BAK or BAX described so far. A body of data supports the existence of core (α2–α5) dimers of BAK and BAX in the oligomeric, membrane-perturbing conformation of these essential apoptotic effector molecules. Molecular structures for these dimers have only been captured for truncated constructs encompassing the core domain alone. Here, we report a crystal structure of BAK α2–α8 dimers (i.e., minus its flexible N-terminal helix and membrane-anchoring C-terminal segment) that has been obtained through the activation of monomeric BAK with the detergent C12E8. Core dimers are evident, linked through the crystal by contacts via latch (α6–α8) domains. This crystal structure shows activated BAK dimers with the extended latch domain present. Our data provide direct evidence for the conformational change converting BAK from inert monomer to the functional dimer that destroys mitochondrial integrity. This dimer is the smallest functional unit for recombinant BAK or BAX described so far. BAK and BAX are essential effectors of mitochondrial apoptosis (Czabotar et al., 2014Czabotar P.E. Lessene G. Strasser A. Adams J.M. Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy.Nat. Rev. Mol. Cell Biol. 2014; 15: 49-63Crossref PubMed Scopus (2010) Google Scholar; Wei et al., 2001Wei M.C. Zong W.X. Cheng E.H. Lindsten T. Panoutsakopoulou V. Ross A.J. Roth K.A. MacGregor G.R. Thompson C.B. Korsmeyer S.J. Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death.Science. 2001; 292: 727-730Crossref PubMed Scopus (3299) Google Scholar). Cells are committed to apoptotic death when BAK/BAX oligomerize and permeabilize the mitochondrial outer membrane (MOM), releasing cytochrome c (cyt c) into the cytosol that triggers the activation of caspases. Pro-survival members of the BCL-2 family restrain BAK/BAX activity. The BH3-only proteins promote apoptosis either by antagonizing BCL-2 or by agonizing BAK/BAX. Thus, BAK/BAX are believed to display different conformations, whether they be quiescent and monomeric, activated but restrained in a complex with pro-survival BCL-2 family members, or activated and unrestrained as pore-forming oligomers. The conformation of inert BAK/BAX is remarkably similar to that of the pro-survival BCL-2 relatives (Birkinshaw et al., 2019Birkinshaw R.W. Gong J.N. Luo C.S. Lio D. White C.A. Anderson M.A. Blombery P. Lessene G. Majewski I.J. Thijssen R. et al.Structures of BCL-2 in complex with venetoclax reveal the molecular basis of resistance mutations.Nat. Commun. 2019; 10: 2385Crossref PubMed Scopus (81) Google Scholar; Day et al., 2005Day C.L. Chen L. Richardson S.J. Harrison P.J. Huang D.C. Hinds M.G. Solution structure of prosurvival Mcl-1 and characterization of its binding by proapoptotic BH3-only ligands.J. Biol. Chem. 2005; 280: 4738-4744Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar; Moldoveanu et al., 2006Moldoveanu T. Liu Q. Tocilj A. Watson M. Shore G. Gehring K. The X-ray structure of a BAK homodimer reveals an inhibitory zinc binding site.Mol. Cell. 2006; 24: 677-688Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar; Muchmore et al., 1996Muchmore S.W. Sattler M. Liang H. Meadows R.P. Harlan J.E. Yoon H.S. Nettesheim D. Chang B.S. Thompson C.B. Wong S.L. et al.X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death.Nature. 1996; 381: 335-341Crossref PubMed Scopus (1277) Google Scholar; Petros et al., 2001Petros A.M. Medek A. Nettesheim D.G. Kim D.H. Yoon H.S. Swift K. Matayoshi E.D. Oltersdorf T. Fesik S.W. Solution structure of the antiapoptotic protein bcl-2.Proc. Natl. Acad. Sci. USA. 2001; 98: 3012-3017Crossref PubMed Scopus (357) Google Scholar; Suzuki et al., 2000Suzuki M. Youle R.J. Tjandra N. Structure of Bax: coregulation of dimer formation and intracellular localization.Cell. 2000; 103: 645-654Abstract Full Text Full Text PDF PubMed Scopus (885) Google Scholar). Their capacity to function in opposition to BCL-2 pro-survival proteins rests in the metastability of the inert monomeric conformer. In contrast to the high-affinity interactions between BCL-2 (and other pro-survival family members) with BH3-only proteins, encounters between BAK/BAX and BH3-only proteins are transient and induce unfolding of the inert BAK/BAX structure. Consequentially, the BH3 domain of BAK/BAX, located in helix α2 and buried in the inert structure, becomes exposed and may be “intercepted” by engaging with pro-survival BCL-2 family members. If pro-survival proteins are unavailable to restrain them, as for example, when they are otherwise engaged with BH3-only proteins, BAK/BAX proceed down a re-folding pathway. This allows reciprocal interactions between their exposed BH3 domain and a hydrophobic surface groove (formed by helices α3, α4, and α5) to form symmetric “BH3-in-groove” dimers. These proceed to higher-order multimerization and the rupture of the MOM (Brouwer et al., 2014Brouwer J.M. Westphal D. Dewson G. Robin A.Y. Uren R.T. Bartolo R. Thompson G.V. Colman P.M. Kluck R.M. Czabotar P.E. Bak core and latch domains separate during activation, and freed core domains form symmetric homodimers.Mol. Cell. 2014; 55: 938-946Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar; Czabotar et al., 2013Czabotar P.E. Westphal D. Dewson G. Ma S. Hockings C. Fairlie W.D. Lee E.F. Yao S. Robin A.Y. Smith B.J. et al.Bax crystal structures reveal how BH3 domains activate Bax and nucleate its oligomerization to induce apoptosis.Cell. 2013; 152: 519-531Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar; Dewson et al., 2008Dewson G. Kratina T. Sim H.W. Puthalakath H. Adams J.M. Colman P.M. Kluck R.M. To trigger apoptosis, Bak exposes its BH3 domain and homodimerizes via BH3:groove interactions.Mol. Cell. 2008; 30: 369-380Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, Dewson et al., 2012Dewson G. Ma S. Frederick P. Hockings C. Tan I. Kratina T. Kluck R.M. Bax dimerizes via a symmetric BH3:groove interface during apoptosis.Cell Death Differ. 2012; 19: 661-670Crossref PubMed Scopus (133) Google Scholar; Subburaj et al., 2015Subburaj Y. Cosentino K. Axmann M. Pedrueza-Villalmanzo E. Hermann E. Bleicken S. Spatz J. García-Sáez A.J. Bax monomers form dimer units in the membrane that further self-assemble into multiple oligomeric species.Nat. Commun. 2015; 6: 8042Crossref PubMed Scopus (110) Google Scholar). Structures of inert monomeric BAK/BAX have been described without (Moldoveanu et al., 2006Moldoveanu T. Liu Q. Tocilj A. Watson M. Shore G. Gehring K. The X-ray structure of a BAK homodimer reveals an inhibitory zinc binding site.Mol. Cell. 2006; 24: 677-688Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar; Suzuki et al., 2000Suzuki M. Youle R.J. Tjandra N. Structure of Bax: coregulation of dimer formation and intracellular localization.Cell. 2000; 103: 645-654Abstract Full Text Full Text PDF PubMed Scopus (885) Google Scholar) and with BH3 peptides bound (Brouwer et al., 2017Brouwer J.M. Lan P. Cowan A.D. Bernardini J.P. Birkinshaw R.W. van Delft M.F. Sleebs B.E. Robin A.Y. Wardak A. Tan I.K. et al.Conversion of Bim-BH3 from Activator to Inhibitor of Bak through Structure-Based Design.Mol. Cell. 2017; 68: 659-672.e9Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar; Czabotar et al., 2013Czabotar P.E. Westphal D. Dewson G. Ma S. Hockings C. Fairlie W.D. Lee E.F. Yao S. Robin A.Y. Smith B.J. et al.Bax crystal structures reveal how BH3 domains activate Bax and nucleate its oligomerization to induce apoptosis.Cell. 2013; 152: 519-531Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar; Moldoveanu et al., 2013Moldoveanu T. Grace C.R. Llambi F. Nourse A. Fitzgerald P. Gehring K. Kriwacki R.W. Green D.R. BID-induced structural changes in BAK promote apoptosis.Nat. Struct. Mol. Biol. 2013; 20: 589-597Crossref PubMed Scopus (149) Google Scholar). Two of the latter studies pointed to the unfolding event, although indirectly, through the formation of non-physiological dimers. In vitro, BH3 peptides induce BAK/BAX constructs lacking their membrane-anchoring segment to transform into dimers in which their core domains (α2–α5) are swapped with their latch domains (α6–α8) (Brouwer et al., 2014Brouwer J.M. Westphal D. Dewson G. Robin A.Y. Uren R.T. Bartolo R. Thompson G.V. Colman P.M. Kluck R.M. Czabotar P.E. Bak core and latch domains separate during activation, and freed core domains form symmetric homodimers.Mol. Cell. 2014; 55: 938-946Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar; Czabotar et al., 2013Czabotar P.E. Westphal D. Dewson G. Ma S. Hockings C. Fairlie W.D. Lee E.F. Yao S. Robin A.Y. Smith B.J. et al.Bax crystal structures reveal how BH3 domains activate Bax and nucleate its oligomerization to induce apoptosis.Cell. 2013; 152: 519-531Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar; Robin et al., 2015Robin A.Y. Krishna Kumar K. Westphal D. Wardak A.Z. Thompson G.V. Dewson G. Colman P.M. Czabotar P.E. Crystal structure of Bax bound to the BH3 peptide of Bim identifies important contacts for interaction.Cell Death Dis. 2015; 6: e1809Crossref PubMed Scopus (39) Google Scholar). These core/latch dimers resemble a dimer of inert monomers, apart from the crossover segment at the junction of helices α5 and α6. They are not physiologically relevant, as their interfaces are inconsistent with data on BAK/BAX oligomerization during apoptosis (Bleicken et al., 2010Bleicken S. Classen M. Padmavathi P.V. Ishikawa T. Zeth K. Steinhoff H.J. Bordignon E. Molecular details of Bax activation, oligomerization, and membrane insertion.J. Biol. Chem. 2010; 285: 6636-6647Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar; Dewson et al., 2009Dewson G. Kratina T. Czabotar P. Day C.L. Adams J.M. Kluck R.M. Bak activation for apoptosis involves oligomerization of dimers via their alpha6 helices.Mol. Cell. 2009; 36: 696-703Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, Dewson et al., 2012Dewson G. Ma S. Frederick P. Hockings C. Tan I. Kratina T. Kluck R.M. Bax dimerizes via a symmetric BH3:groove interface during apoptosis.Cell Death Differ. 2012; 19: 661-670Crossref PubMed Scopus (133) Google Scholar; Zhang et al., 2010Zhang Z. Zhu W. Lapolla S.M. Miao Y. Shao Y. Falcone M. Boreham D. McFarlane N. Ding J. Johnson A.E. et al.Bax forms an oligomer via separate, yet interdependent, surfaces.J. Biol. Chem. 2010; 285: 17614-17627Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). However, they do suggest that the well-characterized BH3-in-groove symmetric dimer form after separation of the core and latch domains. Numerous studies now support the existence of a BH3-in-groove symmetric dimer in the oligomerized form of BAK/BAX. In structural terms, BH3 is helix α2 and the groove is α3α4α5. Forming such a dimer is readily conceived if the core domains of BAK/BAX separate from the remainder of the molecule. Crystal structures of BAK/BAX truncated segments encompassing only α2–α5, first as fusion proteins with GFP (Brouwer et al., 2014Brouwer J.M. Westphal D. Dewson G. Robin A.Y. Uren R.T. Bartolo R. Thompson G.V. Colman P.M. Kluck R.M. Czabotar P.E. Bak core and latch domains separate during activation, and freed core domains form symmetric homodimers.Mol. Cell. 2014; 55: 938-946Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar; Czabotar et al., 2013Czabotar P.E. Westphal D. Dewson G. Ma S. Hockings C. Fairlie W.D. Lee E.F. Yao S. Robin A.Y. Smith B.J. et al.Bax crystal structures reveal how BH3 domains activate Bax and nucleate its oligomerization to induce apoptosis.Cell. 2013; 152: 519-531Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar; Mandal et al., 2016Mandal T. Shin S. Aluvila S. Chen H.C. Grieve C. Choe J.Y. Cheng E.H. Hustedt E.J. Oh K.J. Assembly of Bak homodimers into higher order homooligomers in the mitochondrial apoptotic pore.Sci. Rep. 2016; 6: 30763Crossref PubMed Scopus (32) Google Scholar), and then alone (Cowan et al., 2020Cowan A.D. Smith N.A. Sandow J.J. Kapp E.A. Rustam Y.H. Murphy J.M. Brouwer J.M. Bernardini J.P. Roy M.J. Wardak A.Z. et al.BAK core dimers bind lipids and can be bridged by them.Nat. Struct. Mol. Biol. 2020; 27: 1024-1031Crossref PubMed Scopus (29) Google Scholar), reveal symmetric dimers of core domains featuring BH3-in-groove interactions. These structures confirm both the chemical crosslinking data that first suggested their existence (Dewson et al., 2008Dewson G. Kratina T. Sim H.W. Puthalakath H. Adams J.M. Colman P.M. Kluck R.M. To trigger apoptosis, Bak exposes its BH3 domain and homodimerizes via BH3:groove interactions.Mol. Cell. 2008; 30: 369-380Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, Dewson et al., 2012Dewson G. Ma S. Frederick P. Hockings C. Tan I. Kratina T. Kluck R.M. Bax dimerizes via a symmetric BH3:groove interface during apoptosis.Cell Death Differ. 2012; 19: 661-670Crossref PubMed Scopus (133) Google Scholar) and subsequent spectroscopic measurements (Bleicken et al., 2010Bleicken S. Classen M. Padmavathi P.V. Ishikawa T. Zeth K. Steinhoff H.J. Bordignon E. Molecular details of Bax activation, oligomerization, and membrane insertion.J. Biol. Chem. 2010; 285: 6636-6647Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). However, these crystal structures fall short of demonstrating that core dimers can form from full-length monomeric BAK/BAX. An oligomer of symmetric dimers requires a second, distinct interface. Biochemical crosslinking (Dewson et al., 2009Dewson G. Kratina T. Czabotar P. Day C.L. Adams J.M. Kluck R.M. Bak activation for apoptosis involves oligomerization of dimers via their alpha6 helices.Mol. Cell. 2009; 36: 696-703Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, Dewson et al., 2012Dewson G. Ma S. Frederick P. Hockings C. Tan I. Kratina T. Kluck R.M. Bax dimerizes via a symmetric BH3:groove interface during apoptosis.Cell Death Differ. 2012; 19: 661-670Crossref PubMed Scopus (133) Google Scholar; Iyer et al., 2015Iyer S. Bell F. Westphal D. Anwari K. Gulbis J. Smith B.J. Dewson G. Kluck R.M. Bak apoptotic pores involve a flexible C-terminal region and juxtaposition of the C-terminal transmembrane domains.Cell Death Differ. 2015; 22: 1665-1675Crossref PubMed Scopus (41) Google Scholar; Ma et al., 2013Ma S. Hockings C. Anwari K. Kratina T. Fennell S. Lazarou M. Ryan M.T. Kluck R.M. Dewson G. Assembly of the Bak apoptotic pore: a critical role for the Bak protein α6 helix in the multimerization of homodimers during apoptosis.J. Biol. Chem. 2013; 288: 26027-26038Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) and spectroscopic (Aluvila et al., 2014Aluvila S. Mandal T. Hustedt E. Fajer P. Choe J.Y. Oh K.J. Organization of the mitochondrial apoptotic BAK pore: oligomerization of the BAK homodimers.J. Biol. Chem. 2014; 289: 2537-2551Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar; Bleicken et al., 2010Bleicken S. Classen M. Padmavathi P.V. Ishikawa T. Zeth K. Steinhoff H.J. Bordignon E. Molecular details of Bax activation, oligomerization, and membrane insertion.J. Biol. Chem. 2010; 285: 6636-6647Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar; Mandal et al., 2016Mandal T. Shin S. Aluvila S. Chen H.C. Grieve C. Choe J.Y. Cheng E.H. Hustedt E.J. Oh K.J. Assembly of Bak homodimers into higher order homooligomers in the mitochondrial apoptotic pore.Sci. Rep. 2016; 6: 30763Crossref PubMed Scopus (32) Google Scholar) studies point toward certain residues being in proximity to each other in the oligomer, but no compelling evidence for this second protein-protein interface has yet emerged. In contrast, other data suggest that oligomers can form on the membrane without a specific secondary protein-protein interface (Uren et al., 2017bUren R.T. O’Hely M. Iyer S. Bartolo R. Shi M.X. Brouwer J.M. Alsop A.E. Dewson G. Kluck R.M. Disordered clusters of Bak dimers rupture mitochondria during apoptosis.eLife. 2017; 6: e19944Crossref PubMed Scopus (41) Google Scholar), and blocking studies failed to reveal a secondary interface necessary for MOM permeabilization (MOMP) (Li et al., 2017Li M.X. Tan I.K.L. Ma S.B. Hockings C. Kratina T. Dengler M.A. Alsop A.E. Kluck R.M. Dewson G. BAK α6 permits activation by BH3-only proteins and homooligomerization via the canonical hydrophobic groove.Proc. Natl. Acad. Sci. USA. 2017; 114: 7629-7634Crossref PubMed Scopus (24) Google Scholar). Most recently, a molecular mechanism for multimerization of BAK dimers by the diacyl membrane lipids has been advanced (Cowan et al., 2020Cowan A.D. Smith N.A. Sandow J.J. Kapp E.A. Rustam Y.H. Murphy J.M. Brouwer J.M. Bernardini J.P. Roy M.J. Wardak A.Z. et al.BAK core dimers bind lipids and can be bridged by them.Nat. Struct. Mol. Biol. 2020; 27: 1024-1031Crossref PubMed Scopus (29) Google Scholar). There is no high-resolution structural evidence that core dimers can form from full-length BAK/BAX constructs. Unlike BAX, wild-type BAK cannot be expressed with its transmembrane segment, so here we have used the construct first deployed in describing the monomeric structure that encompasses helices α1–α8 (Moldoveanu et al., 2006Moldoveanu T. Liu Q. Tocilj A. Watson M. Shore G. Gehring K. The X-ray structure of a BAK homodimer reveals an inhibitory zinc binding site.Mol. Cell. 2006; 24: 677-688Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). We show through our structural and biophysical studies that treatment with the detergent C12E8 results in the formation of core domain dimers with latch domains separated and with α1 loosely associated. These dimers are functional both in liposome and mitochondrial permeabilization assays, and they are the smallest active unit of BAK or BAX yet described. To address BAK activation, we expressed a soluble truncated form of human BAK, residues 22–186 consisting of the α1–α8 helices that form its BCL2-like fold (Figure 1). This construct lacks 22 unstructured amino acids at the N terminus and the C-terminal transmembrane domain and includes a thrombin protease site in the α1–α2 loop to allow removal if required (Figure 1). The soluble inert BAK monomers were combined with the non-ionic detergent C12E8, widely used as a non-denaturing detergent for membrane protein purification at a concentration (5.6 mM) that is 60-fold higher than its critical micelle concentration (CMC, 90 μM). The resultant solution was analyzed by size exclusion chromatography (SEC) in a buffer containing C12E8 at a concentration twice its CMC (186 μM). This resulted in a single peak eluting at 12.7 mL, indicating an increase in size relative to a BAK monomer, which eluted at 16.4 mL (Figure 1A). To confirm BAK stoichiometry, we used multi-angle light scattering (MALS), giving an average molecular mass for the 12.7 mL oligomeric peak of 52 kDa (Figure 1B). Thus, as summarized in Table S1, the BAK monomers have a molecular mass of 18.4 kDa, suggesting that the 12.7 mL peak may contain a BAK dimer (36.8 kDa) associated with a small C12E8 micelle. Typical C12E8 micelles range between 48 and 65 kDa, with C12E8 having a molecular mass of 538.8 kDa and an aggregation number between 90 and 120 (le Maire et al., 2000le Maire M. Champeil P. Moller J.V. Interaction of membrane proteins and lipids with solubilizing detergents.Biochim. Biophys. Acta. 2000; 1508: 86-111Crossref PubMed Scopus (801) Google Scholar). Alternatively, the oligomeric species may be a BAK trimer, predicted at 55.2 kDa, with no detergent associated, or simply a BAK monomer associated with a C12E8 micelle (Table S1). The BAK retention volume was further analyzed by performing SEC on the detergent-activated BAK sample in a buffer with no detergent. This resulted in multiple BAK peaks, with early retention volumes indicating an increase in size (Figure 1A). Comparing SEC peak volumes showed that when no detergent was present, the total BAK reduced—a total peak area of 17 mAu/mL for all 3 peaks with no detergent in the running buffer and 25 mAu/mL for the single peak with detergent (Figure 1A). This indicates that C12E8 detergent was required not only to form the complex but also to stabilize it. Therefore, the MALS is interpreted as a BAK dimer associated with C12E8 detergent as opposed to a detergent-free trimer. This is consistent with the BAK dimer, rather than trimer, status observed in lipid environments (Aluvila et al., 2014Aluvila S. Mandal T. Hustedt E. Fajer P. Choe J.Y. Oh K.J. Organization of the mitochondrial apoptotic BAK pore: oligomerization of the BAK homodimers.J. Biol. Chem. 2014; 289: 2537-2551Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar; Dewson et al., 2008Dewson G. Kratina T. Sim H.W. Puthalakath H. Adams J.M. Colman P.M. Kluck R.M. To trigger apoptosis, Bak exposes its BH3 domain and homodimerizes via BH3:groove interactions.Mol. Cell. 2008; 30: 369-380Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar; Mandal et al., 2016Mandal T. Shin S. Aluvila S. Chen H.C. Grieve C. Choe J.Y. Cheng E.H. Hustedt E.J. Oh K.J. Assembly of Bak homodimers into higher order homooligomers in the mitochondrial apoptotic pore.Sci. Rep. 2016; 6: 30763Crossref PubMed Scopus (32) Google Scholar). The SEC and MALS experiments indicate that BAK forms dimers in C12E8 detergent that require detergent to stabilize the dimer; however, it is not possible to exclude a BAK monomer associated with a C12E8 micelle based on this evidence alone. We also tested the role of the BAK α1 helix in BAK dimers. Upon an apoptotic stimulus, BAK forms dimers and higher oligomers in cells to trigger cell death. In these dimers, the BAK α1 helix dissociates from the protein core, exposing the BH4 domain, with antibodies targeted to this region selectively identifying activated BAK (Alsop et al., 2015Alsop A.E. Fennell S.C. Bartolo R.C. Tan I.K. Dewson G. Kluck R.M. Dissociation of Bak α1 helix from the core and latch domains is required for apoptosis.Nat. Commun. 2015; 6: 6841Crossref PubMed Scopus (40) Google Scholar; Cuconati et al., 2002Cuconati A. Degenhardt K. Sundararajan R. Anschel A. White E. Bak and Bax function to limit adenovirus replication through apoptosis induction.J. Virol. 2002; 76: 4547-4558Crossref PubMed Scopus (69) Google Scholar; Griffiths et al., 1999Griffiths G.J. Dubrez L. Morgan C.P. Jones N.A. Whitehouse J. Corfe B.M. Dive C. Hickman J.A. Cell damage-induced conformational changes of the pro-apoptotic protein Bak in vivo precede the onset of apoptosis.J. Cell Biol. 1999; 144: 903-914Crossref PubMed Scopus (393) Google Scholar). To establish the role of the BAK α1 helix, the BAK C12E8-activated dimers were cleaved with thrombin protease to separate the BAK α1 helix and α1–α2 loop from the α2–α8 (Figure 1C). Incubating BAK with the thrombin protease resulted only in partial cleavage, even after several days’ incubation (Figure 1D). Complete cleavage of BAK was achieved when C12E8 detergent (final concentration 5.6 mM) was added to the solution (Figure 1E), indicating that the detergent had induced a conformational change in BAK to expose the thrombin cleavage site. The resultant complex was purified by SEC, showing a single peak in the 280 nm absorbance eluting with a longer retention than the uncleaved BAK dimers and shorter retention than the BAK monomer peaks (Figures 1A and 1B). SDS-PAGE showed that this peak contained only the BAK α2–α8 core and did not include the α1 helix (Figure 1D). Cleaved C12E8-treated BAK had a single peak on SEC. MALS gave a molecular mass for the peak of 40 kDa (Figure 1B). The mass of the cleaved BAK α2–α8 monomer is 13.6 kDa, with the α1 helix and loop accounting for 4.8 kDa of mass (Table S1). The difference in mass between the uncleaved α1–α8 and cleaved α2–α8 according to the SEC-MALS is 12 kDa. The C12E8 micelle mass is unlikely to change significantly between the experiments. This mass loss of 12 kDa between uncleaved α1–α8 and cleaved α2–α8 is more consistent with the loss of two α1 helices and loops than the loss of a single α1 helix and loop (Table S1). This provides additional evidence that uncleaved BAK α1–α8 and cleaved α2–α8 form dimers associated with C12E8, and not monomers or trimers. These experiments also show BAK changes conformation upon the addition of C12E8 that allows the removal of the BAK α1 helix and α1–α2 loop through cleavage at the designed protease site. Furthermore, the removal of the α1 helix does not destabilize BAK C12E8-activated dimers and is therefore not part of the dimer interface. To confirm that the BAK α1–α8 dimers activated by C12E8 detergent were functionally activated, we used an established in vitro liposome release assay (Brouwer et al., 2014Brouwer J.M. Westphal D. Dewson G. Robin A.Y. Uren R.T. Bartolo R. Thompson G.V. Colman P.M. Kluck R.M. Czabotar P.E. Bak core and latch domains separate during activation, and freed core domains form symmetric homodimers.Mol. Cell. 2014; 55: 938-946Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar; Czabotar et al., 2013Czabotar P.E. Westphal D. Dewson G. Ma S. Hockings C. Fairlie W.D. Lee E.F. Yao S. Robin A.Y. Smith B.J. et al.Bax crystal structures reveal how BH3 domains activate Bax and nucleate its oligomerization to induce apoptosis.Cell. 2013; 152: 519-531Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar). These assays monitor the ability of BAK to permeabilize liposomes. A self-quenching fluorescent dye is trapped within the liposome; then, upon permeabilization by BAK, the dye is released, corresponding with an increase in fluorescence. No increase in fluorescence was observed when monomeric BAK α1–α8 (at 540 nM) was incubated with liposomes with or without the activating protein caspase-8-cleaved Bid (cBID) (Figures 2A and S1). However, BAK α1–α8 C12E8-activated dimers produced near-complete dye release at 30 nM and partial release at 10 nM (Figures 2A and S1). Liposomes were also permeabilized by the BAK α2–α8 C12E8-activated dimers and released more dye than the equivalent BAK α1–α8 dimers (Figures 2A and S1). BAK dimers were purified in a buffer containing C12E8 detergent at 186 μM, twice the CMC of 90 μM (le Maire et al., 2000le Maire M. Champeil P. Moller J.V. Interaction of membrane proteins and lipids with solubilizing detergents.Biochim. Biophys. Acta. 2000; 1508: 86-111Crossref PubMed Scopus (801) Google Scholar). To ensure that the liposome permeabilization was specific to BAK dimers and was not an artifact of the C12E8 detergent, size-exclusion buffer including the C12E8 detergent was diluted equivalent to the BAK samples and added to liposomes. This resulted in a final concentration of 12 μM C12E8, 7.5-fold lower than the CMC, and showed negligible dye release from the liposomes (Figures 2A and S1). In addition, we performed assays to determine an approximate threshold for C12E8 in releasing dye from liposomes (Figure S1). The assays showed that C12E8 caused complete dye release from 46.5 μM and above. Partial dye release was shown at 23.3 μM, 10% relative to complete release from a CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) detergent control (Figure S1). The dye release can therefore be attributed to the BAK dimers rather than to the detergent in these assays. Our BAK constructs lack the α9 helix that anchors monomeric BAK to membranes in cells, potentially explaining why our recombinant monomeric BAK did not permeabilize liposomes even in the presence of cBid (Figures 2A and S1). Expressing recombinant BAK with the α9 transmembrane helix without mutations is a challenge. To overcome this limitation, BAK may be targeted to liposomes using a C-terminal hexa-histidine tag (BAKhis) and nickel-charged nitrilotriacetic acid (NTA) lipid headgroups, facilitating direct BAK interactions with the liposome. In the absence of cBid, the BAKhis monomers do not release dye from the liposomes (Figures 2A and S1). When cBid is included, BAKhis induces dye release, demonstrating that permeabilization has occurred as a result of activation (Figures 2A and S1). BAKhis α1–α8 C12E8-activated dimers also showed dye release (Figures 2A and S1). Removal of the α1 helix did not reduce dye release and, as observed with the untagged construct, release was more potent when it was not present (Figures 2A and S1). To further assess the activity of the BAK C12E8-activated (α2–α8) dimers, we compared liposome activity to BAKhis α2–α5 truncated dimers. This α2–α5 truncated constr" @default.
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• W3150454900 date "2021-05-01" @default.
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• W3150454900 title "Structure of detergent-activated BAK dimers derived from the inert monomer" @default.
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• W3150454900 doi "https://doi.org/10.1016/j.molcel.2021.03.014" @default.
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