The interaction of the Bax C-terminal domain with negatively charged lipids modifies the secondary structure and changes its way of insertion into membranes

Ausili, Alessio; Torrecillas, Alejandro; Martinez-Senac, Maria M.; Corbalán-Garcı́a, Senena; Gómez‐Fernández, Juan C.

doi:10.1016/j.jsb.2008.07.004

Cited by 18 publications

(15 citation statements)

References 56 publications

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“…5,57,59,60 The Bak TMD may, however, be non-helical at the carboxy terminus, as partial IASD labeling of four sequential residues is unlikely for a helical TMD. In model membranes, peptides based on the Bak and Bax C termini showed mixtures of secondary structure, 42,44 and the Fis1 C terminus was disordered in NMR studies. 61 Thus, helicity of the TMD may not be necessary for any aspect of Bak function.…”

Section: Discussionmentioning

confidence: 99%

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Bak apoptotic pores involve a flexible C-terminal region and juxtaposition of the C-terminal transmembrane domains

et al. 2015

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Bak and Bax mediate apoptotic cell death by oligomerizing and forming a pore in the mitochondrial outer membrane. Both proteins anchor to the outer membrane via a C-terminal transmembrane domain, although its topology within the apoptotic pore is not known. Cysteine-scanning mutagenesis and hydrophilic labeling confirmed that in healthy mitochondria the Bak α9 segment traverses the outer membrane, with 11 central residues shielded from labeling. After pore formation those residues remained shielded, indicating that α9 does not line a pore. Bak (and Bax) activation allowed linkage of α9 to neighboring α9 segments, identifying an α9:α9 interface in Bak (and Bax) oligomers. Although the linkage pattern along α9 indicated a preferred packing surface, there was no evidence of a dimerization motif. Rather, the interface was invoked in part by Bak conformation change and in part by BH3:groove dimerization. The α9:α9 interaction may constitute a secondary interface in Bak oligomers, as it could link BH3:groove dimers to high-order oligomers. Moreover, as high-order oligomers were generated when α9:α9 linkage in the membrane was combined with α6:α6 linkage on the membrane surface, the α6-α9 region in oligomerized Bak is flexible. These findings provide the first view of Bak carboxy terminus (C terminus) membrane topology within the apoptotic pore. Mitochondrial permeabilization during apoptosis is regulated by the Bcl-2 family of proteins. 1-3 Although the Bcl-2 homology 3 (BH3)-only members such as Bid and Bim trigger apoptosis by binding to other family members, the prosurvival members block apoptosis by sequestering their pro-apoptotic relatives. Two remaining members, Bak and Bax, form the apoptotic pore within the mitochondrial outer membrane (MOM).Bak and Bax are globular proteins comprising nine α-helices. 4,5 They are activated by BH3-only proteins binding to the α2-α5 surface groove, 6-12 or for Bax, to the α1/α6 'rear pocket'. 13 Binding triggers dissociation of the latch domain (α6-α8) from the core domain (α2-α5), together with exposure of N-terminal epitopes and the BH3 domain. 6,7,14-16 The exposed BH3 domain then binds to the hydrophobic groove in another Bak or Bax molecule to generate symmetric homodimers. 6,7,14,17,18 In addition to dimerizing, parts of activated Bak and Bax associate with the lipid bilayer. 19 In Bax, the α5 and α6 helices may insert into the MOM, 20 although recent studies indicate that they lie in-plane on the membrane surface, with the hydrophobic α5 sandwiched between the membrane and a BH3:groove dimer interface. 7,[21][22][23] The dimers can be linked via cysteine residues placed in α6, 18,24,25 and more recently via cysteine residues in either α3 or α5, 6,21 allowing detection of the higherorder oligomers associated with pore formation. 26,27 However, whether these interactions are required for high-order oligomers and pore formation remains unclear.Like most Bcl-2 members, Bak and Bax are targeted to the MOM via a hydrophobic C-terminal region. The C terminus targets Bak to the...

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Section: Discussionmentioning

confidence: 99%

“…The peptides adopt a predominantly α-helical secondary structure, 40-43 with orientation affected by lipid composition. 42,44,45 The peptides could also permeabilize lipid vesicles, 41,43,46,47 suggesting that the …”

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confidence: 99%

Bak apoptotic pores involve a flexible C-terminal region and juxtaposition of the C-terminal transmembrane domains

et al. 2015

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“…The secondary structure components were estimated and the data are reported in Table 1. The results are in a very good agreement with the X-ray data of the protein [22] but to a lesser extent with a previous IR analysis [50] (Table 2), although in this case the secondary structure composition was calculated by using ATR-IR data and possibly the different sample preparation, the use of Lorentzian band shape for the curve-fitting and also the ATR-IR itself, as peaks are known to shift slightly in this technique due to protein adsorption to the crystal, could generate some differences in the secondary structure estimation [51,52]. The protein secondary structure as estimated from our IR data consists of 42% α-helix (including 3 10 -helix), around 25% β-sheet (derived from the sum of all the β-sheet band contributions), around 10% turns (derived from the sum of all the turn band contributions), and 23% unordered structure.…”

Section: This Workmentioning

confidence: 99%

The thermal unfolding of the ribosome-inactivating protein saporin-S6 characterized by infrared spectroscopy

Sánchez¹,

Scirè²,

Tanfani³

et al. 2015

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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“…During membrane pore formation, Bax C-terminus is believed to be α -helical, oriented nearly perpendicular to the membrane plane, stabilized by electrostatic interactions between the two lysines and anionic lipid headgroups. 14,15 Moreover, dominant α -helical structure and transmembrane insertion are thought to occur only in the case of anionic membranes. 14,15 If membrane pores are formed only in anionic membranes by α -helical segments, then efficient carboxyfluorescein release from vesicles made of pure phosphatidylcholine, 12 where the peptide assumes predominantly β -sheet structure, 13,14 remains to be explained.…”

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confidence: 99%

Molecular Basis for Membrane Pore Formation by Bax Protein Carboxyl Terminus

et al. 2012

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Bax protein plays a key role in mitochondrial membrane permeabilization and cytochrome c release upon apoptosis. Our recent data have indicated that the 20-residue C-terminal peptide of Bax (BaxC-KK; VTIFVAGVL-TASLTIWKKMG), when expressed intracellularly, translocates to the mitochondria and exerts lethal effect on cancer cells. Moreover, the BaxC-KK peptide, as well as two mutants where the two lysines are replaced with glutamate (BaxC-EE) or leucine (BaxC-LL), have been shown to form relatively large pores in lipid membranes, composed of up to eight peptide molecules per pore. Here the pore structure is analyzed by polarized Fourier transform infrared, circular dichroism, and fluorescence experiments on the peptides reconstituted in phospholipid membranes. The peptides assume an α/β-type secondary structure within membranes. Both β-strands and α-helices are significantly (by 30–60 deg) tilted relative to the membrane normal. The tryptophan residue embeds into zwitterionic membranes at 8–9 Å from the membrane center. The membrane anionic charge causes a deeper insertion of tryptophan for BaxC-KK and BaxC-LL but not for BaxC-EE. Combined with the pore stoichiometry determined earlier, these structural constraints allow construction of a model of the pore where eight peptide molecules form an “α/β-ring” structure within the membrane. These results identify a strong membranotropic activity of Bax C-terminus and propose a new mechanism by which peptides can efficiently perforate cell membranes. Knowledge on the pore forming mechanism of the peptide may facilitate development of peptide-based therapies to kill cancer or other detrimental cells such as bacteria or fungi.

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The interaction of the Bax C-terminal domain with negatively charged lipids modifies the secondary structure and changes its way of insertion into membranes

Cited by 18 publications

References 56 publications

Bak apoptotic pores involve a flexible C-terminal region and juxtaposition of the C-terminal transmembrane domains

Bak apoptotic pores involve a flexible C-terminal region and juxtaposition of the C-terminal transmembrane domains

The thermal unfolding of the ribosome-inactivating protein saporin-S6 characterized by infrared spectroscopy

Molecular Basis for Membrane Pore Formation by Bax Protein Carboxyl Terminus

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