Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Nov 20;51(46):9406-19.
doi: 10.1021/bi301195f. Epub 2012 Nov 12.

Molecular basis for membrane pore formation by Bax protein carboxyl terminus

Suren A Tatulian  1 , Pranav Garg, Kathleen N Nemec, Bo Chen, Annette R Khaled
Affiliations

Molecular basis for membrane pore formation by Bax protein carboxyl terminus

Suren A Tatulian et al. Biochemistry. .

Abstract

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; VTIFVAGVLTASLTIWKKMG), 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.

PubMed Disclaimer

Conflict of interest statement

Notes

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
The wild-type and mutant peptides reconstituted in lipid membranes adopt an α/β-type secondary structure. ATR-FTIR spectra of BaxC-KK (A, B), BaxC-EE (C, D), and BaxC-LL (E, F) peptides in POPC (A, C, E) or POPC/POPG (7:3) (B, D, F) multilayers deposited on a germanium plate, under an aqueous buffer (150 mM NaCl, 10 mM Hepes in D2O, pH* 6.8). The spectra generated by a linear combination of the original spectra measured at parallel and perpendicular polarizations of the incident infrared light, A = AII + 1.44A⊥, are shown in gray solid lines. The sums of all amide I components are shown in dotted lines. The amide I components corresponding to α-helix, irregular structure (ρ), intramolecular β-sheet, and intermolecular β-sheet are marked by circles, right triangles, inverted triangles, and squares, respectively. Amide I components above 1657 cm−1 shown in solid lines have been assigned to turns. Below each amide I spectrum, its second derivative is shown, in which the downward peaks indicate the locations of amide I components.
Figure 2
Figure 2
The peptides in DPC micelles and lipid vesicles contain α-helical and β-sheet structures. Circular dichroism spectra of BaxC-KK (A, D, G), BaxC-EE (B, E, H), and BaxC-LL (C, F, I) peptides in DPC micelles (A, B, C), POPC unilamellar vesicle membranes (D, E, F) and POPC/POPG (7:3) unilamellar vesicle membranes (G, H, I) at 20 °C (dotted lines) and 37 °C (solid lines). Total concentrations of the lipid, DPC and the peptides were 1.0 mM, 4.0 mM, and 35 μM, respectively, in a buffer containing 20 mM Na,K-phosphate and 0.8 mM EGTA (pH 7.2).
Figure 3
Figure 3
Polarized ATR-FTIR spectroscopy indicates that both α-helices and β-strands are tilted relative to the membrane normal. Polarized ATR-FTIR spectra of BaxC-KK (A, B), BaxC-EE (C, D), and BaxC-LL (E, F) peptides in POPC (A, C, E) or POPC/POPG (7:3) (B, D, F) multilayers deposited on a germanium plate, under an aqueous buffer (150 mM NaCl, 10 mM Hepes in D2O, pH* 6.8). In each panel, spectra measured at parallel and perpendicular polarizations of the infrared light are shown, as indicated. The experimentally obtained spectra are shown in gray solid lines, and the sum of all amide I components in dotted lines. The amide I components corresponding to α-helix, intramolecular β-sheet, and intermolecular β-sheet are marked by circles, inverted triangles, and squares, respectively.
Figure 4
Figure 4
The lipid molecules in supported multilayers are well ordered. ATR-FTIR spectra of POPC/POPG (7:3) multilayers on a germanium plate without (A) and with BaxC-KK peptide (B) at a 1:50 peptide/lipid molar ratio, in the presence of bulk D2O-based buffer (150 mM NaCl, 10 mM Hepes, pH* 6.8), in lipid hydrocarbon chain CH2 stretching region. The estimated thickness of the sample was 1.6 μm. Solid and dashed lines show the spectra at parallel and perpendicular polarizations of the infrared light with respect to the plane of incidence, respectively.
Figure 5
Figure 5
Tryptophan fluorescence of membrane-reconstituted peptides is quenched by brominated lipids. Quenching of tryptophan fluorescence of BaxC-KK (A, B), BaxC-EE (C, D), and BaxC-LL (E, F) by Br2PC lipids brominated at 6,7- (dashed lines), 9,10- (dash-dotted lines), and 11,12-positions (solid lines), at 37 °C. The peptides are reconstituted in POPC (A, C, E) or POPC/POPG (7:3) membranes (B, D, F). Spectra shown in dotted lines correspond to membranes without Br2PC.
Figure 6
Figure 6
The tryptophan of membrane-reconstituted peptides is inserted into the membrane hydrocarbon region. Dependence of quenching of tryptophan fluorescence by brominated lipids on the distance of bromines from membrane center for POPC (A, C) and POPC/POPG (7:3) membranes (B, D), at 37 °C (A, B) and 20 °C (C, D). Circles, triangles, and squares correspond to BaxC-KK, BaxC-EE, and BaxC-LL peptides, respectively. The bell-shaped curves are simulated based on a Gaussian distribution of tryptophan position along the membrane normal (see Materials and Methods); the peaks of the curves indicate the most probable location of the tryptophan from the membrane center.
Figure 7
Figure 7
Structural data allow construction of the pore model. Schematic depiction of the pore formed by eight peptide molecules, four of which are shown in a strand–turn–helix conformation, presented as blue arrow, gray arc, and pink cylinder, respectively. Each peptide molecule is oriented relative to its neighbors in an antiparallel sense. The strands are tilted from the pore central axis by β = 30–40 degrees. The repeat distance of the pore structure, i.e., the distance between ith and (i − 2)th strands, perpendicular to the strand axes, is d.
Figure 8
Figure 8
The molecular model of the pore contains eight peptide molecules and allows efficient transport of calcein. Model for the membrane pore formed by BaxC-KK peptide. Left: Peptide molecules are shown in ribbon format, colored according to peptide monomer. The secondary structure, orientation of strands and helices, and tryptophan insertion into the membrane are based on polarized ATR-FTIR and fluorescence quenching data. Tryptophan 16 and lysines 17 and 18 in each peptide molecule are presented in ball and stick format colored yellow and blue, respectively. Four lipid molecules are shown in ball and stick format, colored according to atom type (carbons gray, oxygens red, hydrogens white, phosphorus orange). Right: Top view of the pore formed by BaxC-KK is shown in a CPK format, colored according to peptide monomer. A calcein molecule is shown within the pore in a ball and stick format, colored according to atom type (see above). Hydrogen atoms in peptide and calcein molecules are omitted.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Youle RJ, Strasser A. The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol. 2008;9:47–59. - PubMed
    1. Tait SW, Green DR. Mitochondria and cell death: outer membrane permeabilization and beyond. Nat Rev Mol Cell Biol. 2010;11:621–632. - PubMed
    1. Westphal D, Dewson G, Czabotar PE, Kluck RM. Molecular biology of Bax and Bak activation and action. Biochim Biophys Acta. 2011;1813:521–531. - PubMed
    1. Suzuki M, Youle RJ, Tjandra N. Structure of Bax: coregulation of dimer formation and intracellular localization. Cell. 2000;103:645–654. - PubMed
    1. Wolter KG, Hsu YT, Smith CL, Nechushtan A, Xi XG, Youle RJ. Movement of Bax from the cytosol to mitochondria during apoptosis. J Cell Biol. 1997;139:1281–1292. - PMC - PubMed

Publication types

MeSH terms

Substances