Crystal Structure of Rat Bcl-xL

Bcl-xL is a member of the Bcl-2 protein family, which regulates apoptosis. Preparation of recombinant rat Bcl-xL yielded two forms, one deamidated at -Asn-Gly- sequences to produce isoaspartates and the other not deamidated. The crystal structures of the two forms show that they both adopt an essentially identical backbone structure which resembles the fold of human Bcl-xL: three layers of two α-helices each, capped at one end by two short helices. Both forms have a long disordered region, which contains the potential deamidation sites. The molecular structure exhibits a low level of interhelical interactions, the presence of three cavities, and a notable hydrophobic cleft surrounded by walls rich in basic residues. These unique structural features may be favorable for its accommodation into membranes or for possible rearrangement to modulate homo-/heterodimerization. Homology modeling of Bcl-2 and Bax, based on the Bcl-xL structure, suggests that Bax has the strongest potential for membrane insertion. Furthermore, we found a possible interface for interaction with non-Bcl-2 family member proteins, such as CED-4 homologues.

The actual functions of the Bcl-2 family proteins are controversial at the molecular level. It has been proposed that homo-/ heterodimerization within the family members (8,20) and with other proteins (21)(22)(23) would be a key event in apoptosis regulation. Yin et al. (15) proposed a hypothesis that Bcl-2 indirectly represses apoptosis through the heterodimerization with Bax to antagonize it. An NMR study revealed the molecular basis for heterodimer formation of Bcl-x L with the peptides derived from Bak (24), whereas mutational analyses showed that the heterodimerization with Bax is not necessarily required for the anti-apoptotic activity of Bcl-x L (25,26). Furthermore, it has been shown that both Bcl-x L and its Caenorhabditis elegans homologue CED-9 bind to CED-4 (21)(22)(23) which is a non-Bcl-2 family apoptosis regulatory protein (27,28).
Recently, Muchmore et al. have reported the three-dimensional structure of human Bcl-x L , and they concluded from the analogy to channel forming proteins, such as the diphtheria toxin, that Bcl-x L also may have an ion channel forming ability (29). Actually, it has been reported that Bcl-x L can form an ion channel in synthetic lipid membranes (30). Thus, the channel formation by Bcl-2 family proteins appears to be another possible mechanism for apoptosis regulation. However, the structure of human Bcl-x L has been solved using the sample prepared through denaturing and refolding processes (29). Therefore, it is important to confirm whether or not a homologous protein more mildly prepared shows a similar conformation to that reported previously.
Using the multiple isomorphous replacement method, we have independently determined the 2.5-Å resolution crystal structure of a recombinant rat Bcl-x L soluble region, which was prepared without denaturing and refolding. It was already established that asparagine residues in the conformationally flexible peptides, especially at the sequence of -Asn-Gly-, are frequently deamidated through a succinimide intermediate formation to yield a mixture of aspartate and isoaspartate (backbone peptide bonds were linked through the side chain ␤carboxyl group of Asp) residues (31)(32)(33)(34). In this report, we demonstrate that rat Bcl-x L indeed can be post-translationally modified by such deamidation, which is facilitated by denaturing process. In addition, homology modeling on the basis of the present crystal structure allowed us to expand the channel formation ability to discussion including the other Bcl-2 family members.

EXPERIMENTAL PROCEDURES
Expression and Purification-cDNA clones for the rat bcl-x L gene were isolated as reported before (11). The DNA fragment encoding the soluble region from Met 1 to Gly 196 was amplified by polymerase chain * This work was supported in part by the Sakabe project of the TARA (Tsukuba Advanced Research Alliance) center at the University of Tsukuba, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The reaction and was introduced into the NcoI and HindIII site of the expression vector pAR2154 (a kind gift from Prof. Y. Nishimura, Yokohama City University), under the control of the T7 RNA polymerase promoter. The plasmid was transfected into Escherichia coli BL21(DE3) by electroporation. The transfectant was grown in LB medium containing a high concentration of ampicillin (200 mg/liter) at 1.0 OD 600 and the Bcl-x L protein was overexpressed by adding isopropyl-1-thio-␤-Dgalactopyranoside to 0.4 mM, followed by continuous culture at 37°C for 12 h with a vigorous shaking. The cells were harvested by centrifugation and disrupted by sonication in a lysis buffer containing 50 mM potassium phosphate, 125 mM potassium chloride, 1 mM EDTA, 25% (v/v) glycerol, 5 mM ␤-mercaptoethanol, 0.2% (v/v) Nonidet P-40, and 0.1 mM phenylmethylsulfonyl fluoride, at pH 6.0. The crude extract was clarified by centrifugation at 140,000 ϫ g for 10 min. Solid fine ammonium sulfate was added to the supernatant to adjust the concentration to 1 M and the resulting precipitate was again removed by centrifugation. Then, the protein fraction containing the expressed Bcl-x L was applied to a hydrophobic column (butyl-Toyopearl, Tosoh) and developed with a linear gradient from 0.5 to 0 M ammonium sulfate. After a step of ultrafiltration, the concentrated sample of Bcl-x L was purified by gel filtration chromatography (Superdex 200, Pharmacia Biotech Inc.). Furthermore, anion exchange column (Mono Q, Pharmacia) chromatography was employed to separate the two forms, designated as Bcl-x L (A) and Bcl-x L (B).
Isoaspartate Analysis-The amount of isoaspartate in proteins or peptides was measured by the ISOQUANT TM protein deamidation detection kit (Promega). Lysylendopeptidase (Wako Pure Chemical) digested peptides were prepared by treatment with 2% (w/w) enzyme at 37°C for 16 h, and were fractionated by reverse phase high performance liquid chromatography (Cosmosil TM 5 C 18 , Nacalai tesque). Analyses of Amino Acid Sequence, Molecular Mass, and Metal Ion-N-terminal amino acid sequences were analyzed with a peptide sequencer (model 492A, Applied Biosystems). MALDI-TOF MS 1 was carried out on a Voyager-Elite TM mass spectrometer (PerSeptive Biosystems), using sinapinic acid (Sigma) as a matrix and horse myoglobin (Sigma) as an external standard. The positive ion was detected in a linear mode. Metal ions (Ca 2ϩ , Zn 2ϩ , Mn 2ϩ , Mg 2ϩ , Cu 2ϩ , Fe 2ϩ,3ϩ , Ni 2ϩ , and Al 3ϩ ) were measured using a Zeeman atomic absorption spectrophotometer (Z-8270, Hitachi) with a graphite furnace.
Crystallization-Crystals were grown at 20°C, by the vapor diffusion method in hanging drops containing 10 l of 7 mg/ml Bcl-x L (B) in 50 mM MES, 5% (v/v) glycerol, 1 mM dithiothreitol, 100 mM NaCl, and 0.7 M Na 2 SO 4 at pH 5.6. The reservoir solution contained 100 mM MES, 10% (v/v) glycerol, 2 mM dithiothreitol, and 1.4 M Na 2 SO 4 at pH 5.6. Oily precipitation appeared within 1 day, and the crystals grew slowly over 3 to 5 days. For the Bcl-x L (A) protein, 1.5 M Na 2 SO 4 was required for crystallization. Before data collection, the crystals were transferred to a harvest buffer containing 1.5 M Na 2 SO 4 , 20% (v/v) glycerol, 1 mM dithiothreitol, and 100 mM MES at pH 5.6. We were unable to crystallize either form using polyethylene glycol 4,000 as a precipitant at pH 7.4, although it was successfully used to produce human Bcl-x L crystals (29).
X-ray Data Collection-Intensity data were collected using either an automated oscillation camera system (DIP-100, DIP-2030, MAC Science) with CuK␣ radiation, generated by a rotating anode M18X (Mac Science), or the macromolecular-oriented Weissenberg camera (35) installed on beamline BL-6A at the Photon Factory of the National Laboratory for High Energy Physics, Tsukuba, Japan. Diffraction data processing was performed with the use of DENZO and SCALEPACK programs (36). All of the diffraction experiments were performed at room temperature.
Structure Determination-Two heavy atom derivatives of Bcl-x L (B) were prepared by soaking the crystals in the harvest buffer lacking dithiothreitol and containing either 5 mM K 2 PtCl 4 or 1 mM EMTS for 1 day. Heavy atom parameters were refined, and the phase calculations were carried out using the MLPHARE (37) program. The multiple isomorphous replacement phases were calculated to 2.8-Å resolution and were improved using either the DM (37) or the SOLOMON (37) program. The X-FIT program in QUANTA (Molecular Simulations Inc.) was used to identify the major ␣-helices in the solvent-flattened electron density map. The phases calculated from the initial structure were combined with the multiple isomorphous replacement phase at 50 to 2.8-Å resolution using SIGMAA (37) and REFMAC (38) programs. After several rounds of model building and phase combining, the structure was refined using the X-PLOR (39) and REFMAC programs. The iterative use of both programs was essential to accomplish the refinement. The final model of Bcl-x L (B), including 145 residues (Ser 2 -Val 30 , Ile 81 -Gly 196 ) and 51 water molecules, was refined to a crystallographic Rfactor of 22.2% (R-free factor ϭ 27.3%), using all reflections between 20.0-and 2.5-Å resolution, by the REFMAC program. The crystal structure of Bcl-x L (A) was initially solved using the rigid-body refinement in the X-PLOR program with Bcl-x L (B) as a starting model, and was refined at 20.0 to 2.8-Å resolution by the X-PLOR and REFMAC programs. The quality of the structure was assessed using the PROCHECK (40) program. The structure determination, the stereochemical statistics, and qualities of the final models are summarized in Table I.

RESULTS
Preparation-The recombinant rat Bcl-x L protein eluted in two separate fractions by anion exchange column chromatography (Fig. 1a). These two proteins showed significantly different migrations by SDS-PAGE analysis (Fig. 1b). However, sequence analysis revealed that both proteins have the same N-terminal sequence, in which the first formylmethionine is processed out by an endogenous E. coli enzyme (41). In addition, the molecular mass analysis by MALDI-TOF MS revealed nearly identical values of 21,936 Ϯ 4 Da and 21,938 Ϯ 2 Da for the peak A and B proteins, respectively; they agree well with the calculated molecular mass of 21,932 Da for the soluble portion of rat Bcl-x L (Ser 2 -Gly 196 ). Analysis by Zeeman atomic absorption spectrometry failed to detect any particular metals bound to either of the proteins, implying that the difference cannot be ascribed to the chelation of metals. Circular dichroism measurements revealed identical helical contents (data not shown). Thus, the two forms of rat Bcl-x L appear to have somewhat different electrostatic properties, although they are identical in their primary and secondary structures. We designated these two forms as Bcl-x L (A) and Bcl-x L (B), respectively.
Isoaspartate Analysis-The rat Bcl-x L protein contains three -Asn-Gly-sequences, which are susceptible to asparagine deamidation (31)(32)(33)(34). The deamidated asparagine then spontaneously converts to either isoaspartate or aspartate. We analyzed the isoaspartate contents in the two forms (Table II); Bcl-x L (B) contained 0.4 mol of isoaspartate/mol of protein, while no isoaspartates were detected in Bcl-x L (A). On the premise that the anion exchange chromatography initially separated the unmodified protein and the deamidated protein into peaks A and B, respectively, the Bcl-x L (B) sample is a mixture of isoaspartate and aspartate forms at one or more of the potential deamidation sites. Bcl-x L (A) is not modified and contains asparagine residues at each site.
To clarify the deamidation site(s), the lysylendopeptidasedigested fragments separated by high performance liquid chromatography were analyzed. In Bcl-x L (B), isoaspartate was found only on the Gly 21 -Lys 87 fragment, which contains two possible deamidation sites (Asn 52 and Asn 66 ); no isoaspartate was detected within the fragment containing the third potential deamidation site (Asn 185 ). However, the present analyses cannot determine which or if both residue(s) (Asn 52 and/or Asn 66 ) was deamidated. Furthermore, the denaturation treatment caused additional deamidation at both Gly 21 -Lys 87 and Glu 158 -Gly 196 fragments, suggesting that all the three -Asn-Gly-sites (Asn 52 , Asn 66 , and Asn 185 ) were deamidated (Table II).
Crystallization and X-ray Crystallography-Crystals of both forms were grown from sodium sulfate solutions by the hanging-drop method. Rat Bcl-x L (A) crystals were usually twinned or clustered, while Bcl-x L (B) reproducibly formed single crystals that diffracted beyond 2.5-Å resolution. The intensity distributions are significantly different between the two forms; R iso for the F data in 45 to 2.8-Å resolution was 13%. Both of the diffraction patterns exhibit thermal diffuse scattering and no-tably large overall temperature factors (64.0 Å 2 for Bcl-x L (A), 55.6 Å 2 for Bcl-x L (B)), indicating a high content of disordered regions.
Molecular Structure-The ribbon drawing of the rat Bclx L (B) molecule is shown in Fig. 2, where we designate the "top" and the "bottom" of the molecule. Bcl-x L (B) consists of eight ␣-helices that form a three-layered all ␣ structure which is similar to those of diphtheria toxin and colicin as originally pointed out by Muchmore et al. (29). The two central ␣-helices, ␣5 and ␣6, are sandwiched between the two layered structures, which consist of the ␣1 and ␣2 helices on one side and the ␣3 and ␣4 helices on the other side. Two ␣ helices, ␣6 and ␣7, appear to be split from a longer helix by a sharp kink. The C-terminal helices, ␣7 and ␣8, are located at the top of the molecule, and they are connected by a turn with the invariant sequence -Asn-Gly-Gly-Trp-.
Helices ␣1 and ␣2 are connected by a long disordered region between Glu 31 and Val 80 . A definitive model for this region could not be built, because of the weak and fragmented electron densities arising from structural disorder in the crystal. The deamidation site(s) of Bcl-x L (B) is/are located within this disordered region.
The overall structure of Bcl-x L (A) were essentially identical to that of Bcl-x L (B); the root mean square deviations between the two models are 0.50 and 0.98 Å for main chain atoms and for all non-hydrogen atoms, respectively. However, notable conformational differences were observed at the loop region connecting ␣2 and ␣3 and both ends of the disordered region. In addition, the analysis of superposition between the two molecules by LSQKAB (37) showed that the spatial arrangements of their main chain atoms are significantly different; the rotation was 0.52°about the axis at the molecular centroid and parallel to the c-axis, and the translations were 0.08 Å, Ϫ0.07 Å, and 0.09 Å about a, b, and c axis, respectively. Thus, the observed intensity difference between the two forms should be due to these contributions.
Comparison of Intramolecular Interactions-Although the intrahelical polar interactions in Bcl-x L (B) are distributed evenly throughout the whole protein, the interhelical interactions are highly localized (Table III). Half of the interhelical interactions are observed between helices ␣1 and ␣2, indicating tight binding. Other regions of the protein have fewer interhelical polar interactions, implying a generally unrestricted tertiary structure. Distribution of non-gly non-Pro / angles in Ramanchandran plot 118 residues (91.5%) in the most favored regions 10 residues (7.8%) in additionally allowed regions 1 residues (0.8%) in generously allowed regions 0 residues (0.0%) in disallowed regions where F pH and F p are the derivative and native (B) structure factor amplitudes, respectively. c Phasing power ϭ RMS(ԽF H Խ/E), where F H is the heavy atom structure factor amplitude, and E is the residual lack of closure error. d R-factor ϭ ⌺ԽԽF p Խ Ϫ ԽF c ԽԽ/⌺ԽF p Խ ϫ 100.
Three hydrophobic cavities were found around the central helices (Fig. 3). One cavity (A) is located in the crossing area between helices ␣2 and ␣5. The second cavity (B) is located in the crossing area between helices ␣1 and ␣5. The third cavity (C) is located between the central helices and the loop connecting helices ␣3 and ␣4. These structural features indicate that the packing around the central helices is relatively loose and unstable (43).
Molecular Surface-The molecular surface of Bcl-x L is predominantly negative, which is consistent with the calculated pI value of 4.4. However, the surface exhibits a remarkably biased charge distribution, mainly due to the concentration of basic residues in the bottom region, whereas acidic residues are distributed uniformly. Furthermore, the molecule exhibits three notable hydrophobic areas, which were also observed in human Bcl-x L . The first area, formed by helices ␣2 (BH3), ␣3, and ␣5 (BH1), is the binding site for the Bak peptide, which has been discussed sufficiently in previous reports (24,29). The second, designated as the bottom cleft, is located at the bottom of the molecule (Fig. 4a), and is created by the C-terminal region of helix ␣5 and the loop connecting helices ␣2 and ␣3. The third, designated as the BH groove, corresponds to the BH1 and BH2 regions in the primary sequence and is located near the top of the molecule. The side chains of two highly conserved tryptophans (Trp 137 and Trp 181 ) are exposed in the central area of this groove.
Comparison with Other Bcl-2 Family Proteins-Since conserved structural features may reveal additional functional information, we built homology models of Bcl-2 and Bax from the Bcl-x L (B) structure, with the amino acid sequence alignment reported before (29). Interestingly, the major features of the molecule, such as the hydrophobic clefts and the biased charge distributions, are well conserved among the three mol-ecules. However, the Bax model appears to exhibit more hydrophobic patches. In particular, the bottom hydrophobic cleft of Bax is relatively broad, and the central helices are more extensively exposed as compared with those of Bcl-x L (Fig. 4b).
Another significant difference is in the hydrogen bonding near the bottom cleft. In Bcl-x L , the central helices are stabilized by two hydrogen bonds ( Fig. 4a and Table III). Notably, these residues are not conserved in Bax (Tyr 22 to Gly 40 , Asp 156 to Cys 126 , Arg 165 to Thr 135 , and deletion of Pro 116 ), suggesting that this region is less restrained in Bax.
Comparison with Human Bcl-x L -Substantial differences are observed in the primary structure between the rat and human Bcl-x L proteins used for x-ray analyses. Human Bcl-x L has 5 extra amino acids at the N terminus and 21 extra residues including polyhistidine tag at the C terminus. In addition, there are five substitutions from rat to human (Glu 40 to Gly 40 , Pro 43 to Ser 43 , Arg 45 to Met 45 , Ser 168 to Ala 168 , and Asp 193 to Glu 193 , respectively). However, the overall three-dimensional structures are almost identical between rat and human Bcl-x L , as revealed from as low values as 1.11 and 1.62 Å of the root mean square deviations for main chain atoms and all nonhydrogen atoms, respectively. The region from Ser 28 to Val 80 is disordered in human Bcl-x L (29), as observed in rat Bcl-x L . The hydrophobic cavities around the central helices are also present in human Bcl-x L .
Except for the N and C termini, and both ends of the disordered region, the most notable difference is observed in the loop (Tyr 101 -Phe 105 ), which connects ␣2 and ␣3 (Fig. 5). This loop exhibits relatively large temperature factors, indicating the high conformational flexibility. To confirm the difference between human and rat Bcl-x L , we calculated annealed omit map (39) of Bcl-x L (B), in which the regional model bias was eliminated. The model of rat Bcl-x L (B), excluding the coordinates for  the residues (Tyr 101 -Phe 105 ), was subjected to the slow cooling procedure in X-PLOR with the initial temperature of 1,000 K. Subsequently, a 2F o Ϫ F c map was calculated using REFMAC. The map fitted to the model of rat Bcl-x L (B) much better than to that of human Bcl-x L , suggesting that the structural differences of the loop region are real.
Comparison to Other Protein Structures-A structural homology search using the program Dali (45) revealed that Bclx L (B) resembles not only the membrane channel forming toxin group (29) but also enzymes such as the vitamin B12-binding domain of methionine synthase and the tetracycline repressor. These proteins contain small molecules sandwiched inside ␣helical layers; these binding sites correspond to hydrophobic cavities or clefts in Bcl-x L .

DISCUSSION
Biological Significance of Deamidation-Deamidation of proteins can occur in vivo, although the biological importance remains obscure (46). The presence of a devoted enzyme, Lisoaspartyl methyltransferase (47,48), implies that deamidation may constitute a pathway to recognize protein damage and to initiate degradation in vivo, and, in other words, be regarded as a "molecular timer." In fact, rat Bcl-x L is often observed as a triplet in Western blot analyses of tissue samples (49,50); this behavior is analogous to the differential mobility of Bcl-x L (A) and Bcl-x L (B) by SDS-PAGE. This implies that the deamidation of Bcl-x L occurs in vivo, although the possibility of other unidentified modifications cannot be ruled out. It would be interesting to determine if deamidation, particularly at frequently occurring sites within the flexible loop, is related to apoptosis regulation.
Potential Function of BH4 -It has been suggested that BH4 may be involved in controlling the homo-/heterodimerization within the Bcl-2 family members. This conserved region corresponds to helix ␣1, which is important for negative regulation of apoptosis in Bcl-x L and Bcl-2 (51). A polar interaction between Asp 83 of Bak and Arg 139 of Bcl-x L is thought to be critical for heterodimerization (24). The residues (Tyr 15 , Lys 16 , and Gln 19 ) in BH4 mask the side chain of Asp 95 (Table III) in BH3, which corresponds to Asp 83 of Bak. Thus, the side chain of Asp 95 is unable to interact with Arg 139 . This fact may be responsible for the inability of Bcl-x L to homodimerize. However, such a hypothesis is somewhat questionable, since the related aspartate in Bax, which heterodimerizes with Bcl-2, also is potentially masked in the same manner.
Possible  hydrophobic cleft which has been reported as an interface for the Bak peptide (24). The structure of this loop was found to be highly flexible. Judging from the figures of previous works of human Bcl-x L (24,29), we assume that the flexible loop would be rearrang its conformation upon binding; the ␣2 and ␣3 helices in the free form possess 5 and 2 turns, respectively, while those in the complex form change to 6 and 1 turns. This flexible conformation may be adaptable enough to bind various counterparts of the BH3 region of Bcl-2 family proteins, such as Bax, Bak, and Hrk.
Interface for CED-4 -Bcl-x L has been shown to interact with the C. elegans protein CED-4 (23); the binding ability is reduced by replacement of -Val 135 -Asn 136 -Trp 137 -in the BH1 region by -Ala-Ile-Leu-. These residues are located at the center of the BH groove, with Trp 137 packed almost perpendicularly against two other conserved tryptophans, Trp 181 and Trp 188 . Thus, the BH groove, formed by the BH1 and BH2 regions, may be the binding site for mammalian CED-4 homologues. Consequently, this suggests that the BH groove is an interface for heterodimerization with non-Bcl-2 family member proteins, although the possibility cannot be excluded that the substitution causes structural disturbances in other regions.
Potential Membrane Pore Formation-On the other hand, several structural features could explain the membrane integration of Bcl-x L . First, the low level of interhelical polar interactions and the presence of hydrophobic cavities suggest that the protein is loosely packed. Thus, the energy barrier for the drastic structural rearrangement that would be required upon membrane insertion might be reduced. Recently, Elkins et al. (52) proposed that membrane pore formation by colicin is related to the three cavities around the central helices. Interestingly, these three cavities are similar to those in the Bcl-x L molecule. Second, the positive charges concentrated at the bottom of the molecule can electrostatically interact with the negative charges of the membrane surface, as frequently observed with channel-forming proteins (53), suggesting that the bottom surface is the origin for membrane insertion. Third, the bottom cleft, which was surrounded by walls rich in basic residues, can act as an initial hydrophobic interface for the lipid aliphatic chains. Fourth, the two central helices, ␣5 and ␣6, are long enough to span a lipid bilayer. Although only the two ␣-helices are insufficient to make a pore, the oligomerization of Bcl-x L itself or its assembly with other protein factors might allow the channel formation.
Homology modeling of Bax revealed the largest bottom hydrophobic cleft and the loss of hydrogen bonds stabilizing the central helices, suggesting that Bax possesses a greater potential for membrane insertion than either Bcl-2 or Bcl-x L . When Bax induced apoptosis is independent of the interleukin 1␤converting enzyme proteases, it is accompanied by a reduction of the mitochondrial membrane potential (54). On the other  (44). The molecules are viewed in the same direction as in Fig. 3. The blue color represents hydrophilic regions and the yellow represents hydrophobic regions. The two red lines in a indicate the hydrogen-bonds: Tyr 22 -Asp 156 and Arg 165 -Pro 116 . The three-dimensional structure of Bax was constructed by homology modeling based on that of Bcl-x L (B) and by using a previously reported sequence alignment (29). a Atom assignments and residue numbers. Wat, water molecule. b HB, hydrogen bond; SB, salt bridge; ID, indirect hydrogen bond. The intramolecular polar interactions (HB,ID: Ͻ3.2 Å; SB: Ͻ4.0 Å) connecting apart more than 10 residues are listed.
hand, both Bcl-x L and Bcl-2 can prevent the loss of this potential during apoptosis (55). Thus, Bax is likely to form membrane pores, whereas pore formation in vivo by Bcl-x L or Bcl-2 is questioned. Therefore, it is more reasonable to consider that the roles of Bcl-x L and Bcl-2 are to inhibit pore formation of Bax or other pore-forming proteins through heterodimerization.
Conclusion-The present crystallographic study of rat Bcl-x L has provided structural insights into the controversial hypotheses for apoptosis regulation by Bcl-2 family members. The structural features revealed not only the putative ability of Bcl-x L to accommodate into membranes but also a possible interface with CED-4 homologues. More extensive structural studies, combined with cellular and biochemical analyses, will be required to draw a more definite conclusion about the true molecular mechanism of apoptosis. FIG. 5. The structure differences between rat and human Bclx L . The root mean square deviations calculated from main chain atoms were plotted after best superposition of entire molecules. The secondary structures were indicated by horizontal arrows and the positions of five substituted residues were also shown by vertical arrows.