Imate C-positions of the R1s in 5 (residues 123?44), 5-6 loop (residues 145?48) and 6 (residues 149?63) are shown relative to the membrane. Helix 6 was tilted toward the N-terminus by 30?by the depth-fitting analysis (see Supplementary Information Figure S6c). (e) The tilting angles of 5 helices in mouse BGH are shown relative to a hypothetical horizontal plane (dotted line). See also Supplemental Figures S5 and S6. of its -carbon (C) but also on the side chain’s direction relative to the membrane normal vector. For this reason, pairs of residues such as 130R1 and 138R1, 106R1 on 4 and 141R1 on 5 had similar depths despite the differences in the depths of the C atoms (Fig. 4b).Scientific RepoRts | 6:30763 | DOI: 10.1038/srepwww.nature.com/scientificreports/The chemical cross-linking results clearly demonstrated the proximity of the C-termini of 3 and 5 helices between neighboring homodimers in the Bak oligomeric pore formed in the mitochondrial outer membrane (Fig. 2a,f,g), confirming our in vitro study27 and its biological relevance. Very recently, similar results were also observed in oligomeric Bax28, indicating that this `3/5 interface’ is common both in Bak and Bax oligomeric pores. The DEER results also support the existence of this interface (Fig. 3g). Recently, Westphal et al. TirabrutinibMedChemExpress GS-4059 proposed a model of lipidic pore formed by apoptotic Bak oligomers30. In this model, Bak BGHs and 9 helices were assumed to remain on the flat region of the membrane while the helical hairpin, formed by 6 and 7-8 extended helices, was hypothesized to line the central lumen of the lipidic pore in a transmembrane orientation, reaching well beyond the core of the membrane. However, our molecular modeling indicated that the 6-8 helical hairpin with the extended length of 30 ? if it existed, is too short to reach beyond the midpoint of a lipidic pore when it is adsorbed to the surface of a lipidic pore formed in a 45?0 ?thick lipid bilayer. Furthermore, if the hypothesized 6-8 helical hairpin existed on the surface of the lipidic pore lumen, parallel arrangement of the hairpins within the pore lumen would make it difficult for 6 helices to make direct contacts between them, contrary to the cross-linking result with Bak/162C (Fig. 2g) and the short inter-spin distance between 162R1-162R1,’ which is 5-12 ?7. Based on the nitroxide inter-spin distances in Bax, Bleicken et al.32 proposed an alternative model of Bax lipidic pore, where the Bax homodimers `clamp’ the toroidal surface of the lipidic pore as mentioned in the Introduction. They assumed that the transmembrane orientation of helix 9 alternates in the membrane. However, it was Stattic site suggested that 9 helices are associated in a parallel transmembrane (TM) orientation in Bax apoptotic pores28,40. Iyer et al. also suggested that the `9:9 interface’ in Bak pore is formed by parallel association of 9 helices in a transmembrane orientation31. Thus, it’s difficult, if not impossible, to envision that the TM helix of Bax or Bak will switch its orientation during pore formation. Zhang et al. recently suggested that Bax 9 helices line the large lipidic pores formed by Bax28. In case of Bak, a TM sequence was not essential in pore formation33 and its direct contribution to the pore structure was not supported experimentally31. Now, a more detailed working model of the Bak lipidic pore, built on our previous one27, is proposed to resolve the above issues (Fig. 5a). Here, the TM 9 helices are hypothesized to interact.Imate C-positions of the R1s in 5 (residues 123?44), 5-6 loop (residues 145?48) and 6 (residues 149?63) are shown relative to the membrane. Helix 6 was tilted toward the N-terminus by 30?by the depth-fitting analysis (see Supplementary Information Figure S6c). (e) The tilting angles of 5 helices in mouse BGH are shown relative to a hypothetical horizontal plane (dotted line). See also Supplemental Figures S5 and S6. of its -carbon (C) but also on the side chain’s direction relative to the membrane normal vector. For this reason, pairs of residues such as 130R1 and 138R1, 106R1 on 4 and 141R1 on 5 had similar depths despite the differences in the depths of the C atoms (Fig. 4b).Scientific RepoRts | 6:30763 | DOI: 10.1038/srepwww.nature.com/scientificreports/The chemical cross-linking results clearly demonstrated the proximity of the C-termini of 3 and 5 helices between neighboring homodimers in the Bak oligomeric pore formed in the mitochondrial outer membrane (Fig. 2a,f,g), confirming our in vitro study27 and its biological relevance. Very recently, similar results were also observed in oligomeric Bax28, indicating that this `3/5 interface’ is common both in Bak and Bax oligomeric pores. The DEER results also support the existence of this interface (Fig. 3g). Recently, Westphal et al. proposed a model of lipidic pore formed by apoptotic Bak oligomers30. In this model, Bak BGHs and 9 helices were assumed to remain on the flat region of the membrane while the helical hairpin, formed by 6 and 7-8 extended helices, was hypothesized to line the central lumen of the lipidic pore in a transmembrane orientation, reaching well beyond the core of the membrane. However, our molecular modeling indicated that the 6-8 helical hairpin with the extended length of 30 ? if it existed, is too short to reach beyond the midpoint of a lipidic pore when it is adsorbed to the surface of a lipidic pore formed in a 45?0 ?thick lipid bilayer. Furthermore, if the hypothesized 6-8 helical hairpin existed on the surface of the lipidic pore lumen, parallel arrangement of the hairpins within the pore lumen would make it difficult for 6 helices to make direct contacts between them, contrary to the cross-linking result with Bak/162C (Fig. 2g) and the short inter-spin distance between 162R1-162R1,’ which is 5-12 ?7. Based on the nitroxide inter-spin distances in Bax, Bleicken et al.32 proposed an alternative model of Bax lipidic pore, where the Bax homodimers `clamp’ the toroidal surface of the lipidic pore as mentioned in the Introduction. They assumed that the transmembrane orientation of helix 9 alternates in the membrane. However, it was suggested that 9 helices are associated in a parallel transmembrane (TM) orientation in Bax apoptotic pores28,40. Iyer et al. also suggested that the `9:9 interface’ in Bak pore is formed by parallel association of 9 helices in a transmembrane orientation31. Thus, it’s difficult, if not impossible, to envision that the TM helix of Bax or Bak will switch its orientation during pore formation. Zhang et al. recently suggested that Bax 9 helices line the large lipidic pores formed by Bax28. In case of Bak, a TM sequence was not essential in pore formation33 and its direct contribution to the pore structure was not supported experimentally31. Now, a more detailed working model of the Bak lipidic pore, built on our previous one27, is proposed to resolve the above issues (Fig. 5a). Here, the TM 9 helices are hypothesized to interact.