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Structural basis for proapoptotic activation of Bak by the noncanonical BH3-only protein Pxt1 [1]

['Dahwan Lim', 'Disease Target Structure Research Center', 'Korea Research Institute Of Bioscience', 'Biotechnology', 'Daejeon', 'Critical Diseases Diagnostics Convergence Research Center', 'Department Of Biochemistry', 'Chungnam National University', 'So-Hui Choe', 'Aging Convergence Research Center']

Date: 2023-07

Bak is a critical executor of apoptosis belonging to the Bcl-2 protein family. Bak contains a hydrophobic groove where the BH3 domain of proapoptotic Bcl-2 family members can be accommodated, which initiates its activation. Once activated, Bak undergoes a conformational change to oligomerize, which leads to mitochondrial destabilization and the release of cytochrome c into the cytosol and eventual apoptotic cell death. In this study, we investigated the molecular aspects and functional consequences of the interaction between Bak and peroxisomal testis-specific 1 (Pxt1), a noncanonical BH3-only protein exclusively expressed in the testis. Together with various biochemical approaches, this interaction was verified and analyzed at the atomic level by determining the crystal structure of the Bak–Pxt1 BH3 complex. In-depth biochemical and cellular analyses demonstrated that Pxt1 functions as a Bak-activating proapoptotic factor, and its BH3 domain, which mediates direct intermolecular interaction with Bak, plays a critical role in triggering apoptosis. Therefore, this study provides a molecular basis for the Pxt1-mediated novel pathway for the activation of apoptosis and expands our understanding of the cell death signaling coordinated by diverse BH3 domain-containing proteins.

Funding: This study was supported by the National Research Foundation of Korea Grants NRF-2019M3E5D6063955 (to B.K.) and NRF-2020R1C1C1006833 (to J.S.), by the National Research Council of Science and Technology of Korea Grant CRC22011-300 (to J.S.), and by the KRIBB Research Initiative Programs KGM9952314 (to B.K.), which were funded by the Ministry of Science and ICT (MSIT) of Republic of Korea. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: All relevant data are within the paper and its Supporting Information files. The coordinates of Bak bound to the Pxt1 BH3 domain have been deposited in the Protein Data Bank ( https://www.rcsb.org ) with the accession code of 8GSV together with the structure factors. The flow cytometry FCS files have been deposited in the FlowRepository database ( http://flowrepository.org ) with the accession code of FR-FCM-Z6AG.

Pxt1, a male germ cell–specific protein exclusively expressed during spermatogenesis [ 28 ], was reported to contain a BH3 domain and to induce germ cell apoptosis and male mouse infertility upon overexpression [ 29 ]. Recently, we determined the crystal structure of Bcl-xL bound to the BH3 domain of human Pxt1 and revealed that human and mouse Pxt1 differ in terms of amino acid length, BH3 domain composition, and binding ability to Bcl-xL [ 27 ]. Furthermore, Aguilar and colleagues reported that the BH3 domain of Pxt1 can directly interact with Bak and induce its activation, which was verified by detecting liposomal membrane permeabilization and cellular cytochrome c release [ 30 ]. In this study, we present our efforts to unravel the functionality of Pxt1 in apoptosis, which led to the confirmation of a direct interaction between Bak and Pxt1 by a combination of diverse biochemical approaches and the complex structure determination by X-ray crystallography. Subsequent crystal structure-based biochemical and cellular analyses demonstrated that Pxt1 functions as an apoptogenic factor by activating Bak via its BH3 domain-mediated intermolecular association. We demonstrated that Pxt1 not only induces dimerization and activates the liposomal membrane-permeabilizing ability of recombinant Bak, but also causes loss of ΔΨm, release of mitochondrial cytochrome c to the cytosol, and significant promotion of apoptotic cell death in its BH3 domain-dependent manner when expressed in cells. Therefore, this study expands our understanding of apoptosis pathways, in which not only conventional Bcl-2 family proteins but also noncanonical members also participate.

B cell lymphoma-2 (Bcl-2) antagonist/killer (Bak) is a critical executor of mitochondrial outer membrane permeabilization (MOMP), the key process of the type I programmed cell death called apoptosis [ 1 – 3 ]. Bak is a member of the Bcl-2 protein family, which comprises 3 subgroups: proapoptotic BH3-only proteins, antiapoptotic Bcl-2 homologues, and downstream MOMP executors Bak and Bcl-2-associated X (Bax) [ 4 , 5 ]. The 3 Bcl-2 family subgroups are intimately associated with each other, which is pivotal for precise regulation of apoptosis. The BH3 domains of Bim, Bid, and Puma, which are called “promiscuous” BH3-only activators, directly interact with the antiapoptotic Bcl-2 homologues [ 6 – 8 ] and also with proapoptotic Bak and Bax [ 9 – 13 ], commonly by being accommodated into a characterized hydrophobic cleft known as the BH3-binding groove. Intermolecular binding of the BH3 domains of Bim, Bid, and Puma initiates the activation of Bak and Bax [ 1 – 3 ]. Once activated, Bak and Bax undergo a conformational change to oligomerize on the mitochondrial membrane, which causes MOMP to be characterized by loss of mitochondrial membrane potential (ΔΨm) and release of cytochrome c [ 14 , 15 ]. The released cytochrome c triggers the assembly of the apoptosome, a large caspase-activating protein complex, which results in consecutive proteolytic cleavage of caspase proteins and eventual apoptotic cell death [ 16 , 17 ]. In contrast, “selective” BH3-only sensitizers, such as Bad and Noxa, interact with antiapoptotic Bcl-2 relatives but not with Bak and Bax [ 7 , 18 – 21 ]. In addition, a variety of proteins, which have not been classified as conventional Bcl-2 family members, contain their own BH3 domains that interact with the BH3-binding groove of antiapoptotic Bcl-2 proteins [ 22 , 23 ]. These proteins include the autophagy regulator Beclin 1 [ 24 ], necrosis regulator SOUL [ 25 ], prosurvival protein TCTP [ 26 ], and apoptosis-associated protein peroxisomal testis-specific 1 (Pxt1) [ 27 ].

Results

Structure determination of Bak in a complex with human Pxt1 BH3 Next, Bak(23–185;C166S) bound to human Pxt1(76–101) was subjected to crystallization, leading to structural determination of this complex to a resolution of 2.2 Å (S1 Table). To the best of our knowledge, this is the first crystal structure of Bak/Bax complexed with the BH3 domain of a noncanonical BH3-only protein. In this complex structure, the human Pxt1 BH3 fragment forms an amphipathic α-helix and is accommodated in the BH3-binding groove of the single Bak molecule (Fig 2A), similar to its binding to the corresponding region of Bcl-xL, with the equivalent positions of the 5 key BH3 consensus residues (S4A Fig). With the lack of detergent treatment during the purification and crystallization steps, C-terminal helix swapping of Bak was not observed in our structure, which was reported to be induced by the combination of detergent and the BH3 fragment and necessary for the 2:2 complex form [9,11,12,34]. The intermolecular binding between Bak and Pxt1 was mainly mediated by hydrophobic interactions: 4 hydrophobic BH3 consensus residues (Leu82, Leu86, Ile89, and Ile93) and 2 additional nonconserved isoleucine residues (Ile78 and Ile79) of human Pxt1 associate with Ile81, Gly82, Ile85, Tyr89, Phe93, Met96, Leu97, Leu100, Tyr110, Lys113, Ile114, Gly126, Val129, Ala130, Leu131, and Phe134 of Bak (Fig 2B). Based on the complex structure, we introduced alanine substitutions at Leu82 and Leu86 in human Pxt1, referred to as DLA in this manuscript, which were expected to abrogate their intermolecular association. A co-immunoprecipitation assay (S2B Fig) and ITC analyses (S3B Fig) demonstrated that these mutations critically impaired complex formation between Bak and human Pxt1, demonstrating the relevance of the crystal structure. In addition, Ile78, Ile79, and Leu82 of human Pxt1, involved in the intermolecular hydrophobic interaction with Bak, are included in the initial part of the α-helix, which is absent in mouse Pxt1 (Figs 2A and S1). Moreover, this region covers approximately 31.0% (367 Å2 of 1,183 Å2) of the total buried surface area of Bak with human Pxt1(76–101) in the complex structure (Fig 2A), which could be the reason that mouse Pxt1 does not interact with Bak or Bax. Consistently, the mouse Pxt1(1–18)-corresponding human Pxt1(84–101) peptide did not show a noticeable association with Bak in ITC analysis (S3B Fig). We also found that Asp91, another conserved BH3 consensus residue in human Pxt1, contributes to complex formation by mediating electrostatic interactions with Arg127 and hydrogen bonding with Asn124 of Bak (Fig 2B). The guanidinium group of Arg87 of Pxt1 also forms a hydrogen bond with the main-chain carbonyl of Ser117 of Bak (Fig 2B). PPT PowerPoint slide

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TIFF original image Download: Fig 2. Structural analysis of the interaction between Bak and Pxt1. (A) Structural representation. Bak is shown in electrostatic surface representation bound to the human Pxt1 BH3 fragment whose side chains are presented as sticks. Human Pxt1 is shown in green, except for the region absent in mouse Pxt1 that is shown in orange. The Pxt1 residues involved in the intermolecular interaction with Bak are labeled. (B) Intermolecular interactions. Pxt1 and Bak are shown in green and gray, respectively. Residues involved in the intermolecular interactions are presented as sticks with labels. Electrostatic interactions and hydrogen bonds between Arg87 and Asp91 of Pxt1 (red) and Ser117, Asn124, and Arg127 of Bak (blue) are marked with dashed lines. (C) Structural rearrangement of Bak induced by Pxt1 binding. Pxt1 is shown in green, while Bak is shown in wheat (apo; PDB code 2IMT) or violet (Pxt1-bound form). The Bak regions undergoing prominent conformational changes are marked by boxes and analyzed in detail. Box I, α3−α4 loop; box II, α2–α3 3 10 -helix and α3 helix; box III, a cavity region in the middle of the BH3-binding groove. The red dot in panel III indicates a water molecule. Bak, Bcl-2 antagonist/killer; Pxt1, peroxisomal testis-specific 1. https://doi.org/10.1371/journal.pbio.3002156.g002

Binding of Pxt1 induces conformational change of Bak Previous structural analyses have revealed that the BH3-binding groove of Bak is closed in the apo form [35,36], suggesting that a conformational change of Bak is inevitable for BH3 domain binding. Thus, we compared our Pxt1 BH3-bound Bak structure to that of apo Bak. Structural superimposition indicated that the 2 structures overlap well with each other with a root mean square deviation value of 1.40 Å over 150 aligned C α atoms. However, severe steric hindrance occurred between the human Pxt1 BH3 fragment and the apo-Bak molecule upon superimposition, in which Ile85, Asn86, Arg88, Tyr89, Glu92, Phe93, and Met96 of Bak were involved (Fig 2C, panel II and S4B Fig). In the complex structure, a 3 10 -helix between α2 and α3 (α2–α3 3 10 -helix; residues 84–88) and the α3 helix (residues 89–100) of Bak containing the steric hindrance-associated residues are moved away toward the opposite side of α4, which widens the BH3-binding groove of Bak and enables the human Pxt1 BH3 domain to be accommodated in the pocket (Figs 2C and S4B). Furthermore, binding of human Pxt1 BH3 induces the formation of a “cavity” in the middle of the BH3-binding groove of Bak (Fig 2C, panel III), which was previously reported to reflect the conformational change of Bak or Bax induced by BH3 domain binding [9,11]. We note that the α3–α4 loop region also undergoes noticeable structural rearrangement upon Pxt1 binding (Fig 2C, panel I).

Structural comparison with other Bak–BH3 complex structures To date, several molecular structures of Bak–BH3 complexes have been reported, including a modified Bid BH3-bound 1:1 form [10], mutated Bid BH3-bound 1:1 form [13], modified Bim BH3-bound 1:1 form, and wild-type Bim BH3-bound 2:2 C-terminal helix-swapped forms [11]. We compared our structure with those of 2 representative Bak–BH3 complexes determined by X-ray crystallography, in which Bak is bound to wild-type Bim BH3, which functions as the Bak activator (2:2 form; PDB code 5VWV), or modified Bim BH3, which serves as the Bak inhibitor (1:1 form; PDB code 5VWZ). As shown in Fig 3A, Pxt1 BH3 binds Bak in nearly the same manner as the other BH3 domains. Noticeably, the Bak region, including the α2–α3 3 10 -helix, α3, and α3–α4 loop, undergoes similar structural rearrangements upon binding of Pxt1 BH3 or the 2 Bim BH3-derived peptides (Fig 3A; also see Fig 2C). Furthermore, the 5 key BH3 consensus residues in the Pxt1 and Bim BH3 domains occupied equivalent positions for binding to the BH3-binding groove of Bak (Fig 3B, left panel). We noted that the cavity region in the middle of the BH3-binding groove of Bak formed upon BH3 binding (see Fig 2C), which is occupied by the nonnatural pentyl-carboxylate group in the Bak-inactivating Bim-h3Pc BH3-bound structure, is empty but for a single water molecule in the Pxt1 or Bim BH3-bound complexes (Fig 3B, right panel; also see Fig 2C, panel III). This implies that Pxt1 BH3 has the potential to trigger a conformational change of Bak to its dimeric form, which is considered to be necessary for Bak activation to provoke apoptosis. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Structural comparison between Bak bound to various BH3 domains. (A) Structural alignments of 3 different BH3 domain-bound Bak molecules. The PDB codes are 5VWV for Bak bound to Bim and 5VWZ for Bak bound to Bim-h3Pc. (B) Details of the intermolecular interactions. Bak in a complex with Pxt1 is shown in an electrostatic surface representation, and 3 peptides accommodated in the hydrophobic groove region are shown together. The 5 BH3 consensus residues are shown as labeled sticks (top) and aligned (bottom). Ф, hydrophobic residue; Σ, small residue; Z, acidic residue; Γ, hydrophilic residue. The cavity region in the middle of the BH3-binding groove of Bak is marked by a rectangle (left) and shown in a separate box (right). (C) Structural comparison of intramolecular electrostatic networks of Bak in the apo or indicated BH3-bound forms. The 4 Bak residues mainly involved in the network are shown as labeled sticks. Dashed lines represent electrostatic interactions. The PDB codes are 2IMT for apo Bak, 7M5A for Bak bound to W3W5-mutant Bid BH3, 7M5B for Bak bound to M3W5-mutant Bid BH3, and 7M5C for Bak bound to Bak BH3. Bak, Bcl-2 antagonist/killer; Pxt1, peroxisomal testis-specific 1. https://doi.org/10.1371/journal.pbio.3002156.g003 Recently, a study done by Singh and colleagues demonstrated that destabilization of the intramolecular electrostatic network of Bak, which is mainly constituted by Arg42, Glu46, Asp90, and Arg137, serves as a hallmark of BAK activation [13]. Therefore, we analyzed the state of the electrostatic network in the Bak–Pxt1 BH3 structure. This network was tightly organized in the apo or Bak-inactivating W3W5-mutant Bid BH3-bound form of Bak (Fig 3C, top panels). In contrast, because of the movement of the Bak α3 helix (see Fig 2C, panel II) containing Asp90 and the side chain reorientation of Arg42, Glu46, and Arg137, the network was considerably destabilized in Pxt1 BH3-bound Bak, similar to that in the Bak-activating M3W5-mutant Bid BH3 or Bak BH3-bound form (Fig 3C, bottom panels). This structural analysis supports the notion that Pxt1 activates Bak via direct binding.

Pxt1 is able to activate recombinant Bak Whether Pxt1 indeed triggers the dimerization and activation of Bak was our next question. To answer this issue, we incubated recombinant Bak with Bim/Pxt1 BH3 at a 1:10 molar ratio in the presence or absence of 1% CHAPS for 1 h and subjected the samples to native gel electrophoresis. Fig 4A shows that new bands with a distinctly increased molecular weight appeared when Bak was incubated with Bim/Pxt1 BH3 in the presence of CHAPS (lanes 4 and 6) but not when either BH3 (lane 2) or CHAPS (lane 8) was absent. To further elucidate these results, SEC analysis was performed, which was used by Brouwer and colleagues to demonstrate that the Bim, Bid, and Bak peptides can induce dimerization of Bak upon treatment with CHAPS [11,34]. Similar to previous results, we noticed that a considerable portion of the Bak protein sample was shifted to a new peak region that appeared only in the presence of Pxt1 BH3 and CHAPS, indicating that Pxt1 BH3 can induce dimerization of Bak (Fig 4B). PPT PowerPoint slide

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TIFF original image Download: Fig 4. Pxt1 BH3 induces dimerization and activation of recombinant Bak. (A) Native gel electrophoresis. Recombinant Bak was incubated with the Bim or Pxt1 BH3 peptide with or without CHAPS. Appearance of a novel protein band can be observed in lanes 4 (100 μM Bak + 1 mM Bim BH3 + 1% CHAPS) and 6 (100 μM Bak + 1 mM Pxt1 BH3 + 1% CHAPS). A full gel figure is available in S1 Raw Images. (B) SEC analysis using a Superdex 75 10/300 GL gel filtration column. The elution positions of the standard protein size markers ovalbumin (43 kDa), carbonic anhydrase (29 kDa), and ribonuclease A (13.7 kDa) are indicated by arrowheads. Samples are prepared with the same concentration described in (A). The peak fractions from the mixture elution were analyzed and visualized by SDS-PAGE and Coomassie staining, shown on the left. Red arrows indicate the novel peak portion that appeared upon incubation with 100 μM Bak, 1 mM Pxt1 BH3, and 1% CHAPS. Full gel figures are available in S1 Raw Images. (C) Liposome release assay. Liposomes encapsulating self-quenching fluorescent dye were incubated with recombinant Bak and/or the indicated BH3 peptide. Release was normalized to detergent-solubilized liposomes. Experiments were performed in independent triplicate, and the numerical data are included in S1 Data. Bak, Bcl-2 antagonist/killer; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; PAGE, polyacrylamide gel electrophoresis; Pxt1, peroxisomal testis-specific 1; S, size marker; SDS, sodium dodecyl sulfate; SEC, size-exclusion chromatography. https://doi.org/10.1371/journal.pbio.3002156.g004 Next, we conducted a liposomal permeabilization assay, which has been widely used to analyze the Bak-activating capability of the BH3 domain-containing fragments [11,30,37–39]. In the mitochondrial outer membrane–mimicking minimal model liposome system (see Materials and methods), the Bak-dependent release of liposome-entrapped carboxyfluorescein was remarkably enhanced by treatment with Pxt1 BH3 in the dose-dependent manner, demonstrating the ability of Pxt1 BH3 to activate Bak (Figs 4C and S5). In contrast, the Pxt1 BH3 peptide lost its liposome-permeabilizing activity upon introduction of the DLA mutation (Figs 4C and S5), indicating that Pxt1 activity relies on its binding to Bak. Collectively, these results demonstrate that human Pxt1 can induce dimerization and activation of recombinant Bak via its BH3 domain-mediated direct binding.

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