Dynamic reconfiguration of pro‐apoptotic BAK on membranes
2021; Springer Nature; Volume: 40; Issue: 20 Linguagem: Inglês
10.15252/embj.2020107237
ISSN1460-2075
AutoresJarrod J. Sandow, Iris K. L. Tan, Allan Shuai Huang, Shashank Masaldan, Jonathan P. Bernardini, Ahmad Z. Wardak, Richard W. Birkinshaw, Robert L. Ninnis, Ziyan Liu, Destiny Dalseno, Daisy Lio, Giuseppi Infusini, Peter E. Czabotar, Andrew I. Webb, Grant Dewson,
Tópico(s)Advanced biosensing and bioanalysis techniques
ResumoArticle15 September 2021free access Source DataTransparent process Dynamic reconfiguration of pro-apoptotic BAK on membranes Jarrod J Sandow Jarrod J Sandow orcid.org/0000-0001-5684-8236 The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Department of Medical Biology, The University of Melbourne, Melbourne, Vic., Australia These authors contributed equally to this work as first authors Search for more papers by this author Iris KL Tan Iris KL Tan The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Department of Medical Biology, The University of Melbourne, Melbourne, Vic., Australia These authors contributed equally to this work as first authors Search for more papers by this author Alan S Huang Alan S Huang orcid.org/0000-0002-2542-1616 The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Department of Medical Biology, The University of Melbourne, Melbourne, Vic., Australia These authors contributed equally to this work as second authors Search for more papers by this author Shashank Masaldan Shashank Masaldan orcid.org/0000-0003-4960-4983 The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Department of Medical Biology, The University of Melbourne, Melbourne, Vic., Australia These authors contributed equally to this work as second authors Search for more papers by this author Jonathan P Bernardini Jonathan P Bernardini orcid.org/0000-0002-5767-7624 The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Department of Medical Biology, The University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Ahmad Z Wardak Ahmad Z Wardak The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Search for more papers by this author Richard W Birkinshaw Richard W Birkinshaw The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Department of Medical Biology, The University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Robert L Ninnis Robert L Ninnis The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Department of Medical Biology, The University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Ziyan Liu Ziyan Liu The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Department of Medical Biology, The University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Destiny Dalseno Destiny Dalseno The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Department of Medical Biology, The University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Daisy Lio Daisy Lio The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Search for more papers by this author Giuseppi Infusini Giuseppi Infusini The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Department of Medical Biology, The University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Peter E Czabotar Peter E Czabotar The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Department of Medical Biology, The University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Andrew I Webb Corresponding Author Andrew I Webb [email protected] orcid.org/0000-0001-5061-6995 The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Department of Medical Biology, The University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Grant Dewson Corresponding Author Grant Dewson [email protected] orcid.org/0000-0003-4251-8898 The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Department of Medical Biology, The University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Jarrod J Sandow Jarrod J Sandow orcid.org/0000-0001-5684-8236 The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Department of Medical Biology, The University of Melbourne, Melbourne, Vic., Australia These authors contributed equally to this work as first authors Search for more papers by this author Iris KL Tan Iris KL Tan The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Department of Medical Biology, The University of Melbourne, Melbourne, Vic., Australia These authors contributed equally to this work as first authors Search for more papers by this author Alan S Huang Alan S Huang orcid.org/0000-0002-2542-1616 The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Department of Medical Biology, The University of Melbourne, Melbourne, Vic., Australia These authors contributed equally to this work as second authors Search for more papers by this author Shashank Masaldan Shashank Masaldan orcid.org/0000-0003-4960-4983 The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Department of Medical Biology, The University of Melbourne, Melbourne, Vic., Australia These authors contributed equally to this work as second authors Search for more papers by this author Jonathan P Bernardini Jonathan P Bernardini orcid.org/0000-0002-5767-7624 The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Department of Medical Biology, The University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Ahmad Z Wardak Ahmad Z Wardak The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Search for more papers by this author Richard W Birkinshaw Richard W Birkinshaw The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Department of Medical Biology, The University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Robert L Ninnis Robert L Ninnis The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Department of Medical Biology, The University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Ziyan Liu Ziyan Liu The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Department of Medical Biology, The University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Destiny Dalseno Destiny Dalseno The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Department of Medical Biology, The University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Daisy Lio Daisy Lio The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Search for more papers by this author Giuseppi Infusini Giuseppi Infusini The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Department of Medical Biology, The University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Peter E Czabotar Peter E Czabotar The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Department of Medical Biology, The University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Andrew I Webb Corresponding Author Andrew I Webb [email protected] orcid.org/0000-0001-5061-6995 The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Department of Medical Biology, The University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Grant Dewson Corresponding Author Grant Dewson [email protected] orcid.org/0000-0003-4251-8898 The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia Department of Medical Biology, The University of Melbourne, Melbourne, Vic., Australia Search for more papers by this author Author Information Jarrod J Sandow1,2, Iris KL Tan1,2, Alan S Huang1,2, Shashank Masaldan1,2, Jonathan P Bernardini1,2, Ahmad Z Wardak1, Richard W Birkinshaw1,2, Robert L Ninnis1,2, Ziyan Liu1,2, Destiny Dalseno1,2, Daisy Lio1, Giuseppi Infusini1,2, Peter E Czabotar1,2, Andrew I Webb *,1,2 and Grant Dewson *,1,2 1The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., Australia 2Department of Medical Biology, The University of Melbourne, Melbourne, Vic., Australia *Corresponding author. Tel: +61 393452832; E-mail: [email protected] *Corresponding author. Tel: +61 393452935; E-mail: [email protected] The EMBO Journal (2021)40:e107237https://doi.org/10.15252/embj.2020107237 See also: LE Sperl et al (October 2021) and AM Ojoawo & T Moldoveanu (October 2021) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract BAK and BAX, the effectors of intrinsic apoptosis, each undergo major reconfiguration to an activated conformer that self-associates to damage mitochondria and cause cell death. However, the dynamic structural mechanisms of this reconfiguration in the presence of a membrane have yet to be fully elucidated. To explore the metamorphosis of membrane-bound BAK, we employed hydrogen-deuterium exchange mass spectrometry (HDX-MS). The HDX-MS profile of BAK on liposomes comprising mitochondrial lipids was consistent with known solution structures of inactive BAK. Following activation, HDX-MS resolved major reconfigurations in BAK. Mutagenesis guided by our HDX-MS profiling revealed that the BCL-2 homology (BH) 4 domain maintains the inactive conformation of BAK, and disrupting this domain is sufficient for constitutive BAK activation. Moreover, the entire N-terminal region preceding the BAK oligomerisation domains became disordered post-activation and remained disordered in the activated oligomer. Removal of the disordered N-terminus did not impair, but rather slightly potentiated, BAK-mediated membrane permeabilisation of liposomes and mitochondria. Together, our HDX-MS analyses reveal new insights into the dynamic nature of BAK activation on a membrane, which may provide new opportunities for therapeutic targeting. SYNOPSIS While BAK is a known executioner protein in apoptotic cell death, how it changes conformation to form oligomeric mitochondrial pores to kill cells remains unclear. Hydrogen-deuterium exchange mass spectrometry and site-directed mutagenesis now resolve key events in BAK activation as it occurs on membranes and mitochondria. Hydrogen-deuterium exchange mass spectrometry provides new insight into conformation changes of BAK on a mitochondria-like membrane. The BAK BH4 domain constrains BAK activity in the absence of a death stimulus. During BAK activation and oligomerisation, amino acids preceding the BH3 domain become exposed and disordered. The disordered N-terminus is not required for BAK to permeabilise membranes and mediate cytochrome c release and may rather inhibit it. Introduction BAK and BAX are pro-apoptotic members of the BCL-2 family of proteins. Their expression and apoptotic activity are essential for cells to die in response to numerous apoptotic stimuli including anoikis, growth factor withdrawal and DNA damage (Wei et al, 2001; Czabotar et al, 2014). In a healthy cell, BAK and BAX are predominantly in an inactive conformation. Following an apoptotic stress, interaction with activated or upregulated BH3-only proteins including BIM and caspase 8-cleaved BID (cBID), BAK and BAX become activated undergoing a drastic change in their conformation. This reconfiguration includes exposure of N-terminal and BH3 domain antibody epitopes, and dissociation of the α2-5 ("core") from α6-8 ("latch") domains (Hsu & Youle, 1998; Griffiths et al, 1999; Dewson et al, 2008; Oh et al, 2010; Czabotar et al, 2013; Brouwer et al, 2014). The activated forms then self-associate by reciprocal interaction of the exposed BH3 domain with a hydrophobic groove of a partner molecule forming symmetric "BH3:groove" homodimers (Dewson et al, 2008, 2012; Czabotar et al, 2013; Brouwer et al, 2014; Subburaj et al, 2015; Zhang et al, 2016). These homodimers then multimerise presumably independent of both the BH3 domain and hydrophobic groove to form high molecular weight structures that permeabilise the mitochondrial outer membrane (Qian et al, 2008; Zhang et al, 2010; Bleicken et al, 2014; Subburaj et al, 2015). Ring-like structures of BAX or BAK that may represent the elusive apoptotic pore have been reported in apoptotic cells (Grosse et al, 2016; Salvador-Gallego et al, 2016), whilst recent studies have shown a role for mitochondrial outer membrane lipids in mediating higher order oligomerisation, consistent with previous work indicating that BAX and BAK form lipidic pores (Terrones et al, 2004; Qian et al, 2008; Salvador-Gallego et al, 2016; Cowan et al, 2020). X-ray crystallography has provided important snapshots of BAK and BAX conformations in solution (Moldoveanu et al, 2006; Czabotar et al, 2013; Brouwer et al, 2014; Birkinshaw et al, 2021). In addition, biochemical approaches such as cross-linking, limited proteolysis and spin labelling have provided insight into changes that occur on mitochondria (Annis et al, 2005; Dewson et al, 2008, 2009, 2012; Bleicken et al, 2010, 2014; Oh et al, 2010; Zhang et al, 2010). However, despite these advances, we lack complete understanding of the molecular changes in BAK/BAX conformation that occur upon activation to facilitate their homodimerisation and subsequent multimerisation to higher order structures. The events that lead to BAK and BAX pore formation have eluded characterisation potentially due to their highly dynamic nature and the requirement for a membrane environment. Indeed, lipid-mediated association of dimers to form a lipidic pore or even random aggregation of dimers to cause membrane rupture are proposed mechanisms (Basanez et al, 2002; Terrones et al, 2004; Uren et al, 2017). Resolving the molecular details of these coordinated events in a membrane environment is key to understand their mechanism of action and will expedite efforts to unlock their emerging and clear potential as therapeutic targets (Niu et al, 2017; van Delft et al, 2019). To overcome these barriers to conventional structural biology approaches and to provide new insight into the conformational dynamics of BAK activation as they occur in a membrane, we employed hydrogen-deuterium exchange mass spectrometry (HDX-MS). HDX-MS exploits the exchange of hydrogen at the amide backbone, the rate of which is governed by hydrogen bonding and solvent accessibility and so is determined by protein conformation, protein-protein and protein-lipid interactions. HDX-MS can be performed in a membrane (Wales & Engen, 2006) and has been used to determine the molecular interactions of large membrane complexes to almost single residue resolution (Sticht et al, 2005), whilst revealing subtle and dynamic changes in protein conformation. Thus, HDX-MS represents a potentially powerful approach to characterise BAK activating conformation change in a membrane environment. We found that activation of BAK in membranes in response to cBID involved exposure of the N-terminus consistent with published findings on the exposure of cryptic antibody epitopes in BAK in apoptotic cells (Griffiths et al, 1999, 2001). HDX-MS also revealed that a significant proportion of the BAK N-terminus becomes highly disordered upon its dissociation from the core of the protein. However, the exposed N-terminus of BAK does not contribute to apoptotic pore formation, but rather limits BAK-mediated membrane damage. Furthermore, mapping the exchange data to existing structures of the BAK monomer and BH3:groove homodimer reveal that disruption of key intramolecular interactions involving the conserved BH4 domain residues is sufficient to trigger BAK activation and apoptotic activity. Results Characterisation of BAK oligomerisation on liposomes BAK is normally integrated into the mitochondrial outer membrane in healthy cells via its C-terminal transmembrane anchor (Iyer et al, 2015). Consequently, full-length BAK is unstable as a recombinant protein due to the hydrophobicity of its C-terminal transmembrane domain. To circumvent this problem, we used recombinant mouse BAK with a 6xHis at its C-terminus in lieu of its transmembrane anchor (BAK-6H). BAK-6H was then targeted to liposomes reconstituted with mitochondrial lipids and a Ni-NTA lipid (Fig 1A). Consistent with previous reports with this system, although BAK alone promoted limited liposome permeabilisation, permeabilising activity was promoted by an activating BID BH3 peptide (Fig 1A). BAK efficiently localised to these liposomes and was largely monomeric and inactive in the absence of an activating stimulus, but oligomerised following activation with a BH3 peptide or recombinant cBID to efficiently permeabilise liposomes (Figs 1B and EV1A and B). Figure 1. BAK-6H activation and oligomerisation on liposomes mirrors changes on mitochondria BAK-6H permeabilising activity is exacerbated by cBID. BAK-6H (mBAK∆C21-6H) was loaded at the indicated concentrations onto Ni-NTA liposomes (5 µM) prior to the addition of cBID. The release of carboxyfluorescein was measured over time. Results representative of at least three independent experiments. BAK-6H oligomerises on liposomes induced by cBID. Liposomes were incubated with BAK-6H (150 nM) and the indicated concentration of cBID or a BID BH3 peptide for 60 min. Liposomes were solubilised in digitonin and BAK oligomers were analysed by BN-PAGE. In parallel, samples were tested at endpoint for liposome permeabilisation based on the release of fluorescent dextran, normalised to total fluorescence of detergent permeabilised liposomes. Results representative of three independent experiments. BAK-6H oligomerisation on liposomes is blocked by BCL-XL. Liposomes were incubated with BAK-6H (150 nM) together with cBID (WT, 90 nM) or cBID M97A (90 nM) that has reduced affinity for BCL-XL, in the presence or absence of BCL-XL for 60 min prior to analysis of BAK oligomerisation by BN-PAGE. Results representative of two independent experiments. Liposome permeabilisation was monitored with the indicated combinations of cBID (90 nM), BAK-6H (150 nM) and BCL-XL (1 µM). Data are presented as mean of two independent experiments with percentage release relative to detergent-lysed liposomes at a timepoint when permeabilisation with BAK and cBid was approximately 50% of detergent-lysis maximum. BAK-6H oligomerisation on liposomes involves its BH3 domain. Liposomes were incubated with BAK-6H (150 nM) together with cBID (90 nM). Antibody recognising the BAK BH3 domain (4B5) or α1-α2 loop (7D10), as shown on the structure of BAK (PDB:2IMS), were added (2.4 µg/µl) either prior to (pre) or after (post) incubation with cBID for 60 min. BAK:Ab complex is indicated (arrowhead). 7D10 which recognises the activated form of mBAK (Iyer et al, 2016) bound to and gel-shifted BAK when added either before or after activation with cBID. In contrast, 4B5 failed to appreciably gel-shift inactive BAK (lanes 3 and 5) or BAK that was already activated (lane 6), but gel-shifted BAK when present during cBID activation (lane 4). Liposome permeabilisation was assessed after 20 min incubation with cBID (90 nM), BAK-6H (150 nM) and antibodies 4B5 (2.5 µg) or 7D10 (2.5 µg). Data are presented as mean of two independent experiments with percentage release relative to detergent-lysed liposomes at a timepoint when permeabilisation with BAK and cBid was approximately 50% of detergent-lysis maximum. HDX-MS reveals structural constraints of BAK on a membrane. BAK-6H (150 nM) on liposomes was deuterated for 0, 10s, 60s or 600s and the incorporation of deuterium was assessed by mass spectrometry. Deuteration is expressed relative to a theoretical maximum. BH3 domain (orange), BH4 domain (blue) and hydrophobic surface groove comprising αhelices 3–5 (green) are shown on the structure of BAK (PDB:2IMS). Differential deuteration of BAK in a membrane. Relative deuteration is mapped onto the structure of inactive monomeric BAK (2IMS, Moldoveanu et al, 2006). Source data are available online for this figure. Source Data for Figure 1 [embj2020107237-sup-0002-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. HDX-MS analysis of BAK-6H during oligomerisation on liposomes A, B. Liposomes were incubated with BAK-6H and cBID at the indicated concentration prior to the analysis of BAK oligomerisation on BN-PAGE. C. BAK peptide coverage following combined proteolysis with pepsin and Aspergillus Type XIII protease. D. Example of time-dependent deuteration of BAK peptide. The increase in m/z of a BAK peptide (shown in red in C) due to incorporation with deuterium over time. Source data are available online for this figure. Download figure Download PowerPoint BAK oligomers forming on mitochondria of apoptotic cells are known to require interaction of an exposed BH3 domain with the hydrophobic groove of a partner BAK molecule (Dewson et al, 2008; Czabotar et al, 2014). To test if the pore-forming oligomers observed in liposomes represent these BH3:groove homo-oligomers, we tested for their ability to be blocked by pro-survival protein BCL-XL. Co-incubation with recombinant BCL-XL blocked BAK oligomerisation on liposomes driven by cBID as assessed by BN-PAGE and liposome permeabilisation (Fig 1C). Blockade by BCL-XL was not just due to its binding cBID to inhibit initial BAK activation as BCL-XL could also inhibit oligomerisation of BAK and permeabilisation triggered by cBIDM97A that does not efficiently bind BCL-XL (Lee et al, 2016). Following BAK activation on mitochondria, the BAK antibody 4B5 can bind the exposed BH3 domain to prevent BAK oligomerisation, but the antibody cannot recognise BAK prior to its activation or once it has oligomerised (Dewson et al, 2008). To confirm that the BAK oligomers induced on liposomes involved BH3:groove homodimers, we tested the ability of the 4B5 antibody to bind BAK either when added before or after BAK dimerisation. The 4B5 antibody failed to gel-shift inactive BAK on liposomes (Fig 1D, lane 3), and likewise when added after BAK oligomerisation (Fig 1D). However, 4B5 antibody could bind when it was added during BAK activation and also impaired liposome permeabilisation (Fig 1D). In comparison, the 7D10 antibody, which binds residues in the BAK α1-2 loop (Iyer et al, 2016), gel-shifted BAK when added either during or after induction of BAK oligomerisation and did not block permeabilisation (Fig 1D). Together, these data support that during BAK activation on, and permeabilisation of, liposomes the BH3 domain is transiently exposed, but is then buried in the oligomer, thus mimicking BAK conformation changes that occur upon activation and oligomerisation on mitochondria. BAK adopts an inactive conformation on liposomes revealed by HDX-MS To characterise deuterium uptake in membrane-associated BAK, we first performed a time course of deuterium incorporation of recombinant BAK∆C21-6H targeted to liposomes followed by quenching of exchange at pH 2.5 and 1°C and combined digestion with the acidic proteases pepsin and Type XIII from Aspergillus saitoi. We found that proteolysis by both proteases was necessary for efficient digestion of BAK resulting in near-complete and overlapping peptide signature, although peptides C-terminal of α4 were under-represented (Fig EV1C). Mass spectrometry revealed a time-dependent increase in the mass of peptides due to deuterium incorporation (Fig EV1D). The rate of exchange correlated well with the published structure of inactive human BAK (2IMS, Moldoveanu et al, 2006), with exposed termini and flexible loop regions exchanging more rapidly than structured regions in the hydrophobic core (Fig 1E and F). Interestingly, the BH4 domain was very resistant to exchange compared with flanking residues in the α1, suggesting a potentially important role for these residues in stabilising the inactive conformer of BAK. Additionally, residues in the α6/7/8 were relatively resistant to exchange despite their predicted solvent exposure in the structure of soluble BAK (Fig 1E and F) (Moldoveanu et al, 2006), suggesting that these residues may interface with lipids of the mitochondrial outer membrane. HDX-MS reveals reorganisation of BAK upon activation on membranes Upon incubation with recombinant cBID to induce BAK activation and oligomerisation (Fig 1), HDX-MS revealed significant changes in BAK conformation as indicated by changes in the rate of deuterium exchange (Fig 2A). Following incubation with cBID, there was a marked increase in deuteration of the BAK α1, particularly corresponding to residues in the conserved BH4 domain (Kvansakul et al, 2008), and in the first half of α2 (Fig 2A and B). This is consistent with the reported exposure of the N-terminus and BH3 domain during BAK activation as indicated by antibody binding and limited proteolysis (Griffiths et al, 1999; Dewson et al, 2008). Figure 2. HDX-MS reveals dynamic changes in BAK activated on liposomes BAK-6H on liposomes was activated or not with cBID for 60 min prior to deuteration for 10s, 60s, 600s, or 3,600s. Incorporation of deuterium was assessed by mass spectrometry. Change in deuteration at each time point is expressed relative to BAK on liposomes without cBID. Deuteration of BAK in a membrane compared to inactive BAK is mapped onto the structure of inactive monomeric BAK (2IMS, Moldoveanu et al, 2006). Deuteration of BAK in a membrane compared to inactive BAK is mapped onto the structure of an activated BAK BH3:groove dimer (4U2V, Brouwer et al, 2014). Download figure Download PowerPoint When changes in deuteration were mapped onto the structure of the hBAK α2-5 homodimer (Brouwer et al, 2014; Birkinshaw et al, 2021), the hinge region between the α2 and α3 exhibited reduced exchange that might imply that the region becomes more structured- or is less solvent-exposed due to protein:protein or protein:lipid interactions. However, we have previously shown that labelling of surface-exposed residues (R88 and E92) in this α2/α3 region with 5 kDa PEG-maleimide did not impair the ability of BAK to oligomerise or to mediate MOMP (Li et al, 2017). This argues against this region of the protein forming important interfaces with either protein or lipid. Comparisons of the X-ray structures of inactive BAK with that of a truncated BAK α2-5 homodimer reveals significant reconfiguration of the α2/α3 hinge region as the α2/BH3 domain becomes exposed and binds into the hydrophobic groove of an opposing BAK monomer, forming the activated BAK homodimer (Fig 2C) (Brouwer et al, 2014). The reduced rate of deuterium exchange in this α2/α3 hinge region in activated BAK is consistent with the straightening of the α2/α3 hinge region observed in dimeric BAK (Brouwer et al, 2014). The trend towards reduced exchange along the α4 suggests that this amphipathic helix is less exposed in the activated form of BAK. Although the peptide coverage in central hydrophobic α5 helix was relatively limited, there was no significant change in the deuterium exchange along this helix upon activation. That α4 becomes more buried whilst the α5 remains so is potentially consistent with the interaction of this lipophilic region with the mitochondrial outer membrane in activated and oligomerised BAK (Czabotar et al, 2013; Cowan et al, 2020). Recently, a crystal structure of oligomeric BAK revealed a hexameric configuration of truncated BAK α2-α5 homodimers in solution and identified lipids, including E. coli-derived phosphoethanolamine (PE), that interacted with α5 residues to stabilise dimer:dimer interactions (Cowan et al, 2020). Whilst the hexamer itself was not considered to represent the precise orientation of BAK dimers within larger BAK oligomers on mitochondria, the structure revealed lipids cross-linking between dimers and supported a model of lipid embedded dimers with a cytosol-exposed α2α3α2′α3′ surface (equivalent to the surface on the outside of the hexamer) and a membrane buried α4α5α5′α4′ surface (equivalent to the surface facing the core of the hexamer) (Fig EV2). We mapped our HDX-MS profiles to this hexameric structure (Fig EV2). Helices α2- α3 reside on the exterior of the hexamer consistent with their increased deuterium exchange following activation (Fig EV2). However, residues in helices α4 and α5 that face the core of the hexameric structure did not exhibit increased exchange following activation and oligomerisation of BAK on a membrane (Figs 2A and EV2), suggesting that α4-α5 do not transition to a more solvent-exposed environment and that they do not line a channel in oligomeric BAK. This aligns with the conclusions of Cowan et al that the protein-protein interactions involved in the crystal do not support the biological relevance of the hexamer as an intermediate in a larger BAK pore-forming oligomer (Cowan et al, 2020) and also reports that BAK forms disordered and heterogeneous complexes on mitochondria (Salvador-Gallego et al, 2016; Uren et al, 2017). Whilst our HDX-MS did not reveal an inhibition of exchange that might support discrete interactions of α5 residues with lipid, or an α4α5α5′α4′ interface that is promoted by lipid interaction, our approach of analysing the exchange profiles before or after full activation of BAK woul
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