Molecular Cell
Article Structural Model of Active Bax at the Membrane
Stephanie Bleicken,1,2,3Gunnar Jeschke,4Carolin Stegmueller,1,2,3Raquel Salvador-Gallego,1,2,3
Ana J.Garcı´a-Sa´ez,1,2,3,*and Enrica Bordignon4,5,*
1Max Planck Institute for Intelligent Systems,Heinbergstras3,70569Stuttgart,Germany
2German Cancer Rearch Center,Im Neuenheimer Feld267,69120Heidelberg,Germany
3Interfaculty Institute of Biochemistry,Eberhard Karls University Tu¨bingen,Hoppe-Seyler-Stras4,72076Tu¨bingen,Germany
4Laboratory of Physical Chemistry,ETH Zurich,Vladimir-Prelog-Weg2,8093Zurich,Switzerland
5Fachbereich Physik,Freie Universita¨t Berlin,Arnimallee14,14195Berlin,Germany
*Correspondence:ana.garcia@uni-tuebingen.de(A.J.G.-S.),enrica.bordignon@fu-berlin.de(E.B.)
dx.doi/10.lcel.2014.09.022
SUMMARYmelody是什么
Bax plays a central role in the mitochondrial pathway of apoptosis.Upon activation,cytosolic Bax mono-mers oligomerize on the surface of mitochondria and change conformation concertedly to punch holes into the outer membrane.The subquent relea of cytochrome c initiates cell death.How-ever,the structure of membrane-inrted Bax and its mechanism of action remain largely unknown. Here,we propo a3D model of active Bax at the membrane bad on double electron-electron reso-nance(DEER)spectroscopy in liposomes and iso-lated mitochondria.We show that active Bax is organized at the membrane as asmblies of dimers. In addition to a stable dimerization domain,each monomer contains a moreflexible piercing domain involved in interdimer interactions and pore forma-tion.The most important structural change during Bax activation is the opening of the hairpin formed by helices5and6,which adopts a clamp-like confor-mation central to the mechanism of mitochondrial permeabilization.
archimedesINTRODUCTIONsuper star是什么意思
Bax and Bak are key regulators of apoptosis that mediate the permeabilization of the mitochondrial outer membrane(MOM) and the relea of cytochrome c(Czabotar et al.,2014;Garcı´a-Sa´ez,2012).
Double knockout mice lacking Bax and Bak die mostly during embryo development or shortly after birth(Linds-ten et al.,2000),and the reduced programmed cell death induces vere abnormalities in the few mice that reach adult-hood.Moreover,cells from BaxÀ/ÀBakÀ/Àmice are resistant to most forms of stress-induced apoptosis,including the artificial expression of BH3-only proteins(Wei et al.,2001).Therefore, the two proteins are esntial for the induction of apoptosis downstream of the BH3-only proteins.
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In healthy cells Bax is mainly a cytosolic,globular,and mono-meric protein,which can also be looly bound to the MOM (Schellenberg et al.,2013).The structure of monomeric Bax solved by nuclear magnetic resonance(NMR)shows an a-heli-cal bundle with a central amphipathic hairpin motif surrounded by solvent-expod helices(Suzuki et al.,2000).Upon induc-tion of apoptosis,Bax permanently inrts into the MOM, where it oligomerizes and induces the relea of cytochrome c and other apoptotic factors(Youle and Strasr,2008). Activation is a multistep process induced by the interaction of Bax with an activator BH3only protein(like tBid or Bim) (Edwards et al.,2013;Gavathiotis et al.,2008;Lovell et al., 2008),followed by the relea of helix a9from the BH groove (Bleicken and Zeth,2009;Kim et al.,2009;Suzuki et al., 2000).Then,helices a2to a4build a symmetric dimer interface (Bleicken et al.,2010;Czabotar et al.,2013;Dewson et al., 2012).Once me
mbrane-embedded,veral amino acids in helices a5,a6,and a9are inaccessible to water,suggesting that they become membrane-inrted(Annis et al.,2005; Garcı´a-Sa´ez et al.,2004).Bad on this and on the structural similarities with colicins,the‘‘umbrella’’model was introduced to reprent active Bax in the membrane.This model propos the inrtion of helices a5and a6as a transmembrane hairpin into the lipid bilayer(Annis et al.,2005).However,the structure of full-length Bax in the membrane environment of the MOM remains elusive to date.
Here,we prent a3D model of a Bax dimer embedded in the membrane with a calculated accuracy of8A˚.To build this model, we ud a multilateration approach bad on distance con-straints gained from Q-band double electron-electron resonance (DEER)on spin-labeled Bax variants inrted into large unilamel-lar vesicles mimicking the MOM lipid composition(MOM-LUVs). The model propod here retains the idea of a core and latch domain in active Bax and Bak(Brouwer et al.,2014;Czabotar et al.,2013,2014),but describes the relative arrangement of the helices in the full-length oligomeric Bax at the membrane. We found that the Bax dimer assumes a clamp-like conformation at the membrane via a partial opening of helices a5and a6that is suggested to be central in the mechanism of membrane perme-abilization.The DEER data show that in full-length active Bax the core domain(helices2–5)builds a stable interaction interface with another analogous domain,in line with th
e crystallized trun-cated GFP-fud dimer found by Czabotar et al.(2013)(Protein Data Bank[PDB]:4BDU).Bad on their function in active Bax dimers,we named helices2–5the dimerization domain.Interest-ingly,we found that the helices beyond a5adopt a moreflexible conformation.Due to their structural features in active Bax
dimers at the membrane,which we suggest to be esntial for membrane destabilization,we named helices6–9the piercing domain.DEER performed on lected Bax mutants interacting with isolated mitochondria corroborated the distance informa-tion obtained in MOM-LUVs,which supports the physiological relevance of the structural model propod.
RESULTS
Spin-Labeled Bax Variants Reproduce the NMR Fold of Monomeric Bax
DEER is a powerful technique to extract dipolar interactions,and thus distance distributions,between spin-labeled probes in pro-teins(distance range from1.5to6nm in membrane-embedded proteins)(Jeschke,2012).In order to apply DEER to Bax,we introduced cysteine mutations to engineer singly and doubly spin-labeled variants(Figure1A).In total,we studied42single and double cysteine mutants of full-length Bax labeled with the nitroxide-bad spin label(1-Oxyl-2,2,5,5-tetramethyl-D3-pyr-roline-
3-methyl)Methanethiosulfonate(MTSL).All spin-labeled Bax variants retained membrane-permeabilizing activity bad on calcein relea from LUVs(Figure1B and Figure S1B avail-able online;detailed information is given in Supplemental Infor-mation).Moreover,we checked that Bax cysteine variants were cytosolic in healthy cells and translocated into distinct foci at mitochondria after apoptosis induction(in line with Nechushtan et al.,2001),indicating that the mutants ud for the EPR measurements are functionally active in cells(Figures 1C and S1C).
Finally,the correct fold of all doubly spin-labeled monomeric proteins was monitored by comparing the intramonomeric dis-tances obtained for25doubly spin-labeled mutants in water with tho simulated on the NMR structures(Suzuki et al., 2000)with the MTSL rotamer library approach implemented in the software MMM2013.2(Polyhach et al.,2011).The NMR model8bestfitted our experimental primary DEER traces(Fig-ure S1D).Figure1D shows DEER traces and distance distribu-tions of all spin pairs compared to tho simulated on model8. The statistical analysis of the deviation between experimental mean distances and tho simulated with MMM2013.2on model 8(Figure S1E)gave a root-mean-square deviation(rmsd)of 3.16A˚,clo to the3.5A˚value calculated using a training t of experimental and simulated distances on water soluble pro-teins and membrane proteins(Jeschke,2013).This proves the very good agreement between the experimental
distances ex-tracted from spin-labeled Bax and the NMR structure(e also Supplemental Information).
The Core Helices2–5Build a Stable Dimerization Domain
To study the active,membrane-embedded Bax conformation, we incubated the spin-labeled variants with cBid in the prence of MOM-LUVs,which led to Bax inrtion and oligomerization in the membrane(Bleicken et al.,2010,2013b;Lovell et al.,2008). Figure2shows the distance distributions obtained in mem-brane-embedded Bax compared to tho obtained in the water-soluble conformation(corresponding DEER traces in Fig-ures S2A–S2D).Notably,the doubly labeled variants were spin diluted with unlabeled wild-type Bax(molar ratio1:4)prior to in-cubation with the MOM-LUVs.This allows enrichment of the signal of the intramonomer distance,while suppressing the inter-monomeric distances and maintaining a sufficient signal-to-noi ratio(e Figures S3A–S3C and Supplemental Informa-tion).By analyzing the intramonomeric distance changes from soluble to membrane-embedded Bax,we grouped the data in two ts showing minor(Figures2B and2C)or major(Figures 2D–2G)changes.
In the membrane-embedded conformation,spin pairs within the core helices a2–a5showed minor cha
nges in the mean intra-monomer distances with respect to the monomeric inactive conformation(Figure2C),indicating that the protein’s tertiary structure was mostly retained upon Bax activation.Most dis-tances were monomodal and relatively narrow,typical of stable, well-defined conformation.However,Bax’s quaternary structure was affected,in fact two core domains built a stable dimerization interface,as we detected defined distances between singly labeled mutants of this region(from C55to C126;Figure2H). Considering the functional role of this region of the protein with respect to the C-terminal part(e below),we named helices 2–5the dimerization domain of active Bax.
The distances within a2–a5in membrane-embedded Bax(Fig-ure2C)are compared in Figure3with tho simulated on the X-ray structure of truncated dimeric Bax fud to GFP(Czabotar et al.,2013)(PDB:4BDU).The structure was elongated in silico up to residue126,using helix a5of monomeric Bax(PDB: 1F16,model8).The4.01A˚rmsd between experimental and simulated mean distances(Figure S2E;Supplemental Informa-tion)is clo to the reference value of3.5A˚(Jeschke,2013), which proves that the X-ray structure is a reliable reprentation of the dimerization domain of active full-length Bax,which had been questioned due to the protein truncation and crystallization in abnce of membranes(detailed information on the method-ology is
given in the Supplemental Information). Additionally,we found that the distance between position16 in a1and position62in the BH3domain became broadly dis-tributed upon Bax activation(Figure2G).A broad distance distri-bution was found also for Bax singly labeled at position16 (Figure2H),indicatingflexibility of a1at the membrane(in agree-ment with Bleicken and Zeth,2009;Gavathiotis et al.,2010;Hsu and Youle,1998;Kim et al.,2009;Yethon et al.,2003).
The peak assignment of spin-diluted doubly labeled proteins having one label in the dimerization domain and the other in the piercing domain was complicated due to the broad dis-tance distributions and unavoidable residual intermonomer peaks(Figures2E and2F).To suppress intermonomer dis-tances we ud the spin dilution approach mentioned before. This was combined with the assignment of residual intermono-mer peaks using the experimentally obtained distances on singly labeled proteins.Additionally,to further corroborate the data,we measured spin-diluted doubly labeled mutants in detergent to increa the signal to noi and prolong the dipolar evolution time(Figures S3and S4).Table1prents thefinal distance constraints obtained(e also the Supple-mental Information).
It is to be noted that due to the oligomeric nature of active Bax at the membrane,both intra-and interdimer distances are
Molecular Cell Model of Active Bax
detected with singly spin-labeled Bax variants,therefore their assignment is difficult (Figure 2H).However,in the dimerization domain,the distance distributions were mainly monomodal and using the available information from the truncated and GFP-fud Bax-dimer (Czabotar et al.,2013),we could annotate them without problems.In contrast,for the broadly distributed distances in the C-terminal part of Bax the assignment was only possible in a few cas by performing crosslinking experi-amelia earhart
ments or by analysis of the model obtained solely with doubly labeled proteins,as described later in the text.
Opening of the a 5-a 6Hairpin Asmbles a Flexible Piercing Domain
When one spin label was positioned in the dimerization domain and the cond one in the C-terminal region of Bax,we detected changes of the mean distances up to 2–4nm upon
membrane
Figure 1.Activity and Folding of the Bax Mutants
(A)Cartoon model of inactive Bax (NMR model 8,PDB 1F16)with the location of spin labels (green,C a atoms).Color code of the helices:yellow (a 1),orange (a 2),gold (a 3),pink (a 4),red (a 5),brown (a 6),violet (a 7),blue (a 8),and green (a 9).
(B)Calcein relea assay from LUVs with Bax wild-type and spin-labeled mutants (the positions of the spin labels are indicated below the bars;data normalized to WT Bax (black bar);shown are mean values and SEM (n =6).All mutants are shown in Figures S1A and S1B.
(C)Fixed HeLa cells cotransfected with Mito-DsRed2(magenta)and GFP-Bax S101C;L149C (green,wild-type Bax and additional mutants are shown in Figure S1C).Control conditions compared to 2hr incubation with 1m M staurosporine.Scale bar reprents 20m m.Ints show zooms of the different channels.
(D)Experimental Q-band DEER traces V(t)/V(0)(black,left panels)and distance distributions obtained via Tikhonov regularization with DeerAnalysis2013(Jeschke et al.,2006)(black,right panels).The V(t)/
V(0)traces are the raw DEER data and they show the modulation of the normalized echo intensity versus dipolar evolution time.Superimpod to the experimental time traces and distance distributions,tho simulated with MMM2013.2(Polyhach et al.,2011)on NMR model 8.The average distance distributions simulated using all NMR models are also prented in dotted red (the extracted mean distances are prented in Figure S1E).See also Figure S1.
Molecular Cell
Model of Active Bax
inrtion (Figures 2E and 2F),indicating large rearrangements in the C-terminal region of the protein.As helices a 6to a 9are involved in membrane-inrtion and pore formation (Annis
et al.,2005;Garcı
´a-Sa ´ez et al.,2006;Nechushtan et al.,1999),we refer to this C-terminal half of active Bax as the piercing domain.It is to be noted that helix a 5,which belongs to the dimerization domain is necessary for both Bax dimerization (George et al.,2007)and pore formation (Annis et al.,2005;Gar-cı
´a-Sa ´ez et al.,2006).In the ‘‘umbrella’’model,the central hairpin of helices 5and 6acts as a membrane inrtion domain around which the rest of the protein helices unfold on the membrane surface.
Strikingly,
weightwatchersFigure 2.Conformational Changes Induced by Bax Activation
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(A)Location of the spin-labeled residues in a schematic view of the protein.Color code of the helices and corresponding spin-labeled sites:yellow (a 1),orange (a 2),gold (a 3),pink (a 4),red (a 5),brown (a 6),violet (a 7),blue (a 8),and green (a 9).Site 169,in the loop connecting helices a 8-a 9,is associated with the light green color and the C-terminal site 193with the green color (as for sites in helix a 9).
(B)Model of inactive Bax (PDB:1F16,model 8)with helices 2–5colored according to (A)and the rest of the protein colored in gray.Black lines connect spin-labeled pairs within the core helices 2–5,which show minor distance changes upon Bax activation.
(C)Amplitude-normalized distance distributions in the soluble monomeric (gray)and membrane-embedded oligomeric (black)state in the dimer-ization domain.
(D)Model of inactive Bax (PDB:1F16,model 8)with helices 1and 6–9colored according to (A)and the rest of the protein colored in gray.Lines con-nect spin labels from the dimerization domain to position
s 149(brown lines),169(light green),193–186(green),and 16(yellow),which show major distance changes upon Bax activation.
(E–G)Corresponding amplitude-normalized dis-tance distributions from doubly labeled mutants in the soluble monomeric (gray)and membrane-embedded oligomeric (black)state.Asterisks denote peaks assigned to intermonomer dis-tances (e Figure S4).Red arrows highlight the more relevant distance changes.
(H)Distance distributions in membrane-embedded Bax obtained with singly labeled mu-tants.All DEER traces are shown in Figure S2.The mean distances extracted from the prented distance distributions are shown in Table 1and Figure S2E.
See also Figures S3and S4.
we detected conformational changes incompatible with a persistent hairpin fold when we considered the spin pair connecting the beginning and the end of the hairpin (residues 101and 149).In monomeric Bax,this pair shows a 2.5nm distance fingerprint of the hairpin
fold,but upon Bax activation this distance incread to 5nm (Fig-ure 2E).Moreover,other distances b
etween spin pairs connect-ing the dimerization domain to position 149became longer and broadly distributed upon Bax membrane inrtion (Figure 2E).Therefore,the ‘‘umbrella’’model does not adequately reprent Bax conformation in the membrane.
The recently published structure of a domain-swapped dimer of Bax triggered by detergent (Czabotar et al.,2013)captured an off-pathway full opening of helices 5and 6.We simulated intra-monomer distances between the dimerization domain and position 149or 169in the swapped dimer and found that they remble our experimental DEER distances.However,the
Molecular Cell
Model of Active Bax
intermonomer distances within the swapped dimer are incom-patible with our data (Figure S3D),confirming that the domain-swapped dimer does not reprent the conformation of active membrane-embedded Bax.Notably,by treating soluble Bax with low detergent concentration we obtained experimental hints pointing to a measurable fraction of domain swapped dimer in the sample (Figures S3E and S3F),suggesting that the pres-ence of detergent molecules in low amounts is responsible for the formation of this nonphysiological structure.
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bca
Next,we analyzed the distances between residue 169(begin-ning of C-terminal helix a 9)and the dimerization domain in mem-brane-embedded Bax.The most drastic distance change upon Bax activation (4nm)was found between position 62in helix a 2and position 169(Figure 2E),indicating that the opening of the hairpin a 5–a 6pulled the C-terminal region away from the dimerization domain.A significant distance increa was also detected between 101or 87and 169(Figure 2E).The end of helix a 9was mapped with spin labels at residues 193(C terminus)and 186(end of helix a 9).We obrved that the mean distances to the dimerization domain also incread up to 3nm (e.g.,87–193and 101–193,Figure 2E)upon membrane inrtion,corroborating the idea that the globular fold in aqueous buffer is lost upon Bax activation.The large width of the distance distributions demon-strates that helix a 9in membrane-inrted Bax is structurally dy-namic.This is illustrated by the distances between 62,87,and 186.The 62–87distance in the dimerization domain is narrowly distributed (Figure 2C),but spin pairs containing one of the
two sites paired with 186in helix a 9gave a broad distance dis-tribution (62–186and 87–186,Figure 2E),indicating that the flex-ibility comes from the label in a 9.
Modeling Active Bax at the Membrane
From the collected DEER constraints,we built a structural model of full-length membrane-embedded Bax.As a starting point we ud the X-ray structure of helices a 2–a 5extended up to posi-tion 126,which agrees with our experimental constraints within this domain.We localized the spin labels at positions 149,169,186,and 193with respect to the dimerization domain by applying the nanoscale multilateration process available in the software MMM2013.2(Polyhach et al.,2011).This approach us the basic principles of GPS localization,but adapted to the nitroxide midpoint of spin labels in proteins (e Supple-mental Information ).We validated this multilateration approach on monomeric Bax in water,for which we had six sites con-strained by more than four DEER distances.The highest location probability obtained for each labeled site shows a
4.9A
˚rmsd with respect to the mean location of the nitroxide labels simulated with MMM2013.2on the available NMR struc-ture (model 8)(e Figure S5C and Supplemental Information ).We did the same with the coordinates of three sites (62,87,and 126)localized via multilateration on the crystal structure of the dimerization domain (PDB:4BDU)and found an rmsd
of 7.8A
˚(Figure S5D and Supplemental Information ).For mem-brane-inrted Bax,we therefore consider 8A
˚to be
the Figure 3.The Crystal Structure of the Truncated a 2-5Dimer in Detergent Reprents the Dimeriz
ation Domain of Full-Length Bax at the Membrane
(A)Truncated dimeric structure of Bax (PDB:4BDU)with helix 5elongated up to residue C126using as template the helix of monomeric Bax (PDB:1F16,model 8).As an example the calculated MTSL rotamers at C62in the BH3domains (helices a 2)and at 126at the end of helices a 5are shown in stick reprentation.The colored spheres reprent the population density of the nitroxide radical.
(B)Left,simulated (gray,red)versus experimental (black,as in Figure S2)DEER traces of the singly labeled variants after background correction (F(t)/F(0)).Right,simulated (gray,red)versus experimental (black,as in Figure 2)distance distributions.Color code:gray,pairs in the crystal structure (PDB:4BDU);red:pairs involving C126(in the elongated helix a 5).See also Figures S2and S3.introduce怎么读
Molecular Cell
Model of Active Bax
