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. 2012;7(4):e35910.
doi: 10.1371/journal.pone.0035910. Epub 2012 Apr 23.

Dynamic interaction of cBid with detergents, liposomes and mitochondria

Stephanie Bleicken  1 , Ana J García-Sáez, Elena Conte, Enrica Bordignon
Affiliations

Dynamic interaction of cBid with detergents, liposomes and mitochondria

Stephanie Bleicken et al. PLoS One. 2012.

Abstract

The BH3-only protein Bid plays a key role in the induction of mitochondrial apoptosis, but its mechanism of action is still not completely understood. Here we studied the two main activation events of Bid: Caspase-8 cleavage and interaction with the membrane bilayer. We found a striking reversible behaviour of the dissociation-association events between the Bid fragments p15 and p7. Caspase-8 cleavage does not induce per se separation of the two Bid fragments, which remain in a stable complex resembling the full length Bid. Detergents trigger a complete dissociation, which can be fully reversed by detergent removal in a range of protein concentrations from 100 µM down to 500 nM. Incubation of cBid with cardiolipin-containing liposomes leads to partial dissociation of the complex. Only p15 (tBid) fragments are found at the membrane, while p7 shows no tendency to interact with the bilayer, but complete removal of p7 strongly increases the propensity of tBid to become membrane-associated. Despite the striking structural similarities of inactive Bid and Bax, Bid does not form oligomers and reacts differently in the presence of detergents and membranes, highlighting clear differences in the modes of action of the two proteins. The partial dissociation of cBid triggered by the membrane is suggested to depend on the strong and specific interaction between p15 and p7. The reversible disassembly and re-assembly of the cBid molecules at the membrane was as well proven by EPR using spin labeled cBid in the presence of isolated mitochondria. The observed dynamic dissociation of the two Bid fragments could allow the assistance to the pore-forming Bax to occur repeatedly and may explain the proposed "hit-and-run" mode of action of Bid at the bilayer.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Structural similarities between mouse Bid and human Bax.
A. NMR structure of mouse Bid (PDB 1DDB) with the two natural cysteines spin labeled in this study (C126 in p15 and C30 in p7) and the cleavage site in ball and stick models. B. Superposition of mouse Bid (colored) and human Bax (black, PDB 1F16). Color code for alpha helices in mouse Bid: 1, light yellow; 1/2, dark yellow; 2, orange; 3, gold; 4, pink; 5, red; 6, brown; 7, violet.
Figure 2
Figure 2. Oligomerization and structural stability of Bid.
A. SEC chromatograms (Superdex 75 column) of FL-Bid, cBid and tBid. Black curves, soluble proteins; dark cyan, 2% OG; red, 1% DDM. For tBid the elution profile in the presence of DTT is also shown (dotted lines). B. SDS PAGE of cBid and tBid (before and after spin labeling) in the presence and absence of DTT. C. CD spectra of soluble and detergent-incubated (1% DDM) FL-Bid (black and red, respectively); soluble and detergent-incubated (1% DDM) cBid (gray and orange, respectively). D. Normalized melting curves of the Bid variants. Black and gray, soluble FL-Bid and FL-BidR1, respectively; light and dark green, soluble cBid and cBidR1, respectively; blue, soluble tBid. E. Normalized melting curves of the Bid variants in the presence of 1% DDM. Red, FL-Bid; orange and yellow, cBid and cBidR1, respectively; brown, tBid.
Figure 3
Figure 3. Effect of detergent on Bid structure and dynamics.
Room temperature continuous wave EPR spectra of FL-BidR1 (A) and cBidR1 (B) in the absence (black) and presence of 0.5 (yellow) and 2% DDM (red) and 1 (cyan) and 2% OG (dark cyan). Arrows highlight the immobile (i) and mobile (m) spectral components. C, D. Normalized form factors F(t) and distance distributions obtained by fit of the DEER traces with the software DeerAnalysis2010 in the absence and presence of detergents (color code as in panel A). Protein concentrations 40 µM.
Figure 4
Figure 4. Reversibility of the cBid dissociation at different protein concentrations.
(A) Normalized DEER traces V(t), normalized form factors F(t) and distance distributions obtained with the software DeerAnalysis2010 of cBidR1 in the absence of detergent (A), in the presence of 2% OG (B, only V(t) is presented), and after removal of detergent by extensive dialysis (C). Asterisks denote noise-related artefacts in the distance distribution. Protein concentration 180 µM. (D) Tryptophan and tyrosine (Y47, W48, Y185) fluorescence spectra of cBidR1 detected after excitation at 280 nm in the absence of detergent (black), in the presence of 2% OG (gray), and after removal of detergent by extensive dialysis (dotted black). Protein concentration 0.5 µM.
Figure 5
Figure 5. Bid-membrane interactions.
A. Low field region of the EPR spectra of cBidR1 detected at 37°C in buffer (protein concentration 40 µM, dotted grey) and after 2 minutes incubation (black) of 3.5 µL cBidR1 (40 µM) with 4.5 µL ECL liposomes (lipid concentration 4.5 mg/ml). The arrow highlights the small increase in spin label mobility. B. Spectra of cBidR1 in the presence of ECL liposomes after 2 minutes (black), 3 h (red) and 16 h (green) incubation. C. Intensity normalized spectra of cBidR1 in water (black). Spectra detected in the supernatant fraction (blue) and in the washed pellet fraction (red) after 3 h incubation of 50 µL cBidR1 (110 µM) with 50 µL ECL or MOML liposomes (20 mg/ml lipids) at 37°C. The same results were reproduced with 4 times higher lipid to protein ratio (not shown). D. EPR spectrum of tBidR1 in water (black). Asterisks denote a small fraction of residual unbound MTSSL biradical in solution. Spectra detected in the supernatant fraction (blue) and in the washed pellet fraction (red) after 3 h incubation of 20 µL tBidR1 (50 µM) with with 50 µL MOML liposomes (20 mg/ml lipids). The spectrum in the supernatant is assigned to the residual MTSSL free in solution. E. SDS PAGE of cBidR1 and tBidR1 in the supernatant fraction (sup.) and in the washed pellet fraction (pe.). The left lane shows the reference molecular weights, asterisks denote the liposome bands. Silver staining of this gel showed some cBid in the pellet fraction as well as some tBid in the supernatant fraction (data not shown).
Figure 6
Figure 6. Binding of p15 and p7 to GUVs.
cBidp15red (C30S) and cBidp7red (126S) are shown in (A) and (B), respectively. GUVs composed of 80% phosphatidylcholine (egg), 20% cardiolipin (bovine heart) and <0.05%. DiO (Invitrogen) was incubated with 25 nM of the Alexa 633 labeled cBid variant. DiO is shown in the first panel (green) and the Alexa 633 labeled cBid variant in the second (red). The merge of the red and the green channel is shown in the third panel. The bar indicates 50 µm. Pictures were taken after 1 h. Due to better visualization the brightness in the red channel was increase for cBidp7red (126S). Notably, due to the fragility of the GUVs, 10–20% of the total GUVs are permeabilized even in absence of any protein.
Figure 7
Figure 7. Exchange of Bid molecules at the mitochondrial membrane.
Room temperature continuous wave EPR spectra of cBidR1 in the presence of isolated mitochondria (1.6 mg/ml lipid concentration). The central upper spectrum of cBidR1 is obtained immediately after addition of mitochondria (60 µl sample, 20 µM protein concentration, calculated spin concentration 42 µM). The left column shows the control spectrum after 1 h incubation at 37°C (addition of 20 µl buffer after the first 20 min incubation, spin concentration 32 µM); the spectrum of the pellet resuspended in 10 µl buffer (calculated spin concentration 36 µM) and the spectrum in the supernatant (calculated spin concentration 21 µM). The right column shows the analogous spectra obtained after addition of 14-fold protein excess of competitor unlabeled cBid (addition of 20 µl of cBid 860 µM after the first 20 min incubation, spin concentration 30 µM). The spectrum in the pellet (spin concentration 16 µM) reveals the reduced amount of spin labeled protein at the membrane in the presence of competitor unlabeled protein. In the supernatant the spin concentration was found to be 28 µM.
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