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Originally published In Press as doi:10.1074/jbc.M700492200 on March 2, 2007

J. Biol. Chem., Vol. 282, Issue 17, 13123-13132, April 27, 2007
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Crystal Structure of the Bcl-XL-Beclin 1 Peptide Complex

BECLIN 1 IS A NOVEL BH3-ONLY PROTEIN*

Adam Oberstein, Philip D. Jeffrey, and Yigong Shi1

From the Lewis Thomas Laboratory, Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544

Received for publication, January 17, 2007 , and in revised form, March 2, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Bcl-2 family proteins are key regulators of apoptosis and have recently been shown to modulate autophagy. The tumor suppressor Beclin 1 has been proposed to coordinate both apoptosis and autophagy through direct interaction with anti-apoptotic family members Bcl-2 and/or Bcl-XL. However, the molecular basis for this interaction remains enigmatic. Here we report that Beclin 1 contains a conserved BH3 domain, which is both necessary and sufficient for its interaction with Bcl-XL. We also report the crystal structure of a Beclin BH3 peptide in complex with Bcl-XL at 2.5Å resolution. Reminiscent of previously determined Bcl-XL-BH3 structures, the amphipathic BH3 helix of Beclin 1 bound to a conserved hydrophobic groove of Bcl-XL. These results define Beclin 1 as a novel BH3-only protein, implying that Beclin 1 may have a direct role in initiating apoptotic signaling. We propose that this putative apoptotic function may be linked to the ability of Beclin 1 to suppress tumor formation in mammals.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Programmed cell death (1) is a critical process in metazoans responsible for basic tissue homeostasis, selective elimination of damaged cells, and structural reorganization during development. Apoptosis, the most clearly defined form of programmed cell death, is initiated by an array of intracellular and extracellular signals (e.g. DNA damage, cytokine removal, anoikis, etc.) and is characterized by a canonical morphology exhibiting chromatin condensation, nuclear fragmentation, and membrane blebbing (2, 3). The molecular mechanisms responsible for this morphology involve the release of cytochrome c and other apoptogenic factors from the mitochondria, and the subsequent activation of caspases, a family of cysteine proteases that dismantle the cell in a precise and systematic manner (4-6). The Bcl-2 family of proteins integrate apoptotic signal transduction upstream of and at the mitochondrial membrane (7, 8). Three classes of Bcl-2 family proteins have been extensively described in the literature: anti-apoptotic Bcl-2-like proteins (Bcl-2, Bcl-XL, Bcl-W, and Mcl-1, A1) containing three or four Bcl-2 homology (BH)2 domains; pro-apoptotic Bax-like proteins (Bax, Bak, and Bok) containing BH1, -2, and -3 domains; and pro-apoptotic BH3-only proteins (e.g. Bim, Bad, Bid, PUMA, and Noxa) containing only the BH3 domain. Upon receipt of an apoptotic stimulus, BH3-only proteins are then "activated," repress anti-apoptotic Bcl-2 family members, and activate pro-apoptotic Bax-like proteins via direct protein-protein interaction (9-11). This leads to oligomerization of Bax and Bak at the mitochondrial membrane, release of cytochrome c, and subsequent activation of caspases. Thus, BH3-only proteins form a family of critical cell death ligands, which initiate a canonical death pathway and the resultant apoptotic morphology.

Autophagy, a highly conserved stress response, may function as an alternative, non-apoptotic death mechanism under certain conditions, for which it is sometimes referred to as type II programmed cell death (12-14). Under most conditions, autophagy is thought to function as a catabolic recycling mechanism involving the sequestration of cytoplasm and/or entire organelles into double-membraned vacuoles, termed autophagosomes, and their subsequent delivery to the lytic compartment (15). This type of autophagy, termed macroautophagy, functions to facilitate bioenergetic buffering during metabolic stress and turnover of long-lived proteins and organelles (15). During nutrient limitation, autophagy serves as a survival mechanism, maintaining a reserve of macromolecular building blocks and ATP in both wild-type (16) and transformed (17, 18) cells.

Interestingly, abnormal induction of autophagic vacuoles, a signature of type II programmed cell death, has been observed in dying cells from many diverse species (12, 19), which has led some to believe that autophagy may have a direct role in such a death process. Consistent with this hypothesis, molecular evidence for autophagy-dependent cell death has recently been observed in mouse L929 cells treated with the pan-caspase inhibitor Z-VAD-fmk (20) and apoptosis-deficient bax-/-, bak-/- mouse embryonic fibroblasts treated with etoposide or staurosporine (21). In both cases, RNA interference directed toward components of the general autophagy machinery reverses the death phenotype. These studies bolster the notion that autophagy, an intrinsically self-limiting process, can, at least in some situations, act as a bona fide cell death program.

Additionally, Bcl-2 itself has been shown to directly inhibit autophagic cell death via interaction with the mammalian autophagy gene beclin 1, demonstrating functional cross-talk between the apoptotic and autophagic pathways (22). Beclin 1, the first identified mammalian autophagy protein (23), can function as a tumor suppressor in mammals (24-26) and interacts with the anti-apoptotic Bcl-2 family proteins Bcl-2 and Bcl-XL but not Bax (22, 27, 28). A litany of human cell lines, each expressing a mutant Beclin 1 deficient in binding to Bcl-2, all display increased levels of type II cell death, which is suppressed by small interfering RNA of the essential autophagy gene atg5 (22). These results suggest that Bcl-2, in addition to its hallmark ability to block type I-programmed cell death, can also block type II-programmed cell death, perhaps multiplying its oncogenic potential (22, 29). They also imply that anti-apoptotic Bcl-2 family members, acting upstream of Beclin 1, may serve as master cell death regulators, able to coordinate both apoptotic and autophagic signals.

However, the precise molecular mechanisms underlying the regulation of autophagy by Bcl-2 family proteins remain poorly understood. For example, it is unclear how Beclin 1 interacts with Bcl-XL or Bcl-2. Such lack of mechanistic understanding is further confounded by unpredictable cell biology observations. For example, overexpression of Bcl-2 or Bcl-XL in wild-type mouse embryonic fibroblasts treated with etoposide appears to stimulate, rather than inhibit, Beclin 1-dependent autophagic cell death (21). The cause of this reversal of phenotype in the presence of an apoptotic stimulus remains unclear (22).

To gain further insight into the regulation of Beclin 1 by Bcl-2 family proteins, we have performed biochemical experiments using purified proteins. In this study, we have defined the minimal interaction domains between Bcl-XL and Beclin 1. We have discovered that Beclin 1 contains a conserved BH3 domain and determined the crystal structure of the Beclin 1 BH3 peptide in complex with Bcl-XL. The structure, combined with in vitro binding analysis, reveals that the Beclin 1 BH3 domain is both necessary and sufficient for interaction with the hydrophobic groove of Bcl-XL and proves that Beclin 1 is an authentic BH3-only protein. The classification of Beclin 1 as a BH3-only family member implies that, at least under certain circumstances, Beclin 1 may act upstream of Bcl-2, mediating a cryptic apoptotic signaling function. We speculate that this putative pro-apoptotic function may partially explain the ability of Beclin 1 to suppress tumor formation in mammals.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Expression and Purification of Proteins—The Beclin 1 cDNA was generously provided by Dr. Dan Klionsky (University of Michigan). All constructs were generated using a standard PCR-based cloning strategy and confirmed by sequencing. The Bcl-2 binding domain of human Beclin 1 (Bcl-2BD, residues 86-158) as well the human Beclin 1 BH3 domain (BeclinBH3, residues 107-135) were expressed as N-terminal glutathione S-transferase (GST) fusions from the vector pGEX-2T (Pharmacia Corporation). Human Bcl-XL{Delta}loop{Delta}TM (deletion of residues 27-82 and the C-terminal 24 residues) was cloned into pRSF (Novagen) and coexpressed with GST-BeclinBH3 in Escherichia coli strain BL21 (DE3) at 15 °C. The soluble fraction of the E. coli lysate was passed over a glutathione-Sepharose 4B (Amersham Biosciences) affinity column, and the BeclinBH3-Bcl-XL complex was cleaved from the column using thrombin. The complex was then further purified using ion exchange (Source 15Q, Amersham Biosciences) and size exclusion (Superdex-200, Amersham Biosciences) chromatography. Wild-type and mutant GST-Bcl-2BD were expressed individually at room temperature and purified using a similar purification procedure, except that intact GST fusions were eluted from the affinity resin using reduced glutathione. A variant of Bcl-XL{Delta}TM (N52D,N66D) was expressed as a C-terminal His6 fusion from pET29b (Novagen) and purified to homogeneity using affinity (nickel-nitrilotriacetic acid, Qiagen), ion exchange (Source 15Q), and size exclusion (Superdex-200) chromatography.

Crystallization and Data Collection—BeclinBH3-Bcl-XL co-crystals were grown by hanging drop vapor diffusion at 22 °C by mixing equal volumes of protein (5 mg/ml) with a well buffer containing 19-24% polyethylene glycol 400, 0.1 M HEPES, pH 7.5, and 0.2 M tri-sodium citrate dihydrate. Crystals appeared overnight and were harvested after 10 days of maturation. The crystals belong to the space group P21212 and contain two domain-swapped dimers (four complexes) per asymmetric unit. The unit cell dimensions are a = 107.94 Å, b = 110.59 Å, and c = 100.52 Å. Crystals were equilibrated in a cryoprotectant buffer containing 15% glycerol (v/v) plus well buffer and flash frozen in a -170 °C nitrogen stream. Native data were collected at National Synchrotron Light Source beamline X-25 and processed using Denzo and Scalepack software (30).

Structural Determination—The structure was determined by molecular replacement using the program PHASER (31) and coordinates from the Bim-Bcl-XL crystal structure (accession code 1PQ1), excluding the Bim peptide. The atomic model was built using O (32) and refined using crystallography NMR software (33). Weak non-crystallography symmetry restraints (5-15 kcal/mol) were used in refinement. Electron density for the Beclin 1 peptide and the domain-swapped helices were unambiguous after preliminary refinement. The final, refined atomic model contains residues 107-127 of human Beclin 1 and residues 2-198 of human Bcl-XL with amino acids 26 and 83 contiguous. The Beclin 1 peptide contains two additional N-terminal residues (Gly and Ser) carried over from the thrombin cleavage site of pGEX-2T.

GST-mediated Pulldown Assay—1 mg of wild-type or mutant GST-Bcl-2BD protein was immobilized on a 0.3-ml glutathione S-Sepharose 4B column and washed with 20 column volumes of buffer A (25 mM Tris, pH 8.0, 150 mM NaCl, 3 mM dithiothreitol) to remove unbound protein. 2 mg of Bcl-XL{Delta}TM was then bound to the resin followed by two 10-column volume washes with buffer A. Bound proteins were eluted with buffer A plus 5 mM reduced glutathione, applied to SDS-PAGE, and visualized by staining with Coomassie Blue.

Isothermal Titration Calorimetry (ITC)—Wild-type or mutant Bcl-2BD or a synthetic peptide identical to the BeclinBH3 peptide used for crystallization was titrated into the reaction cell of a VP-ITC microcalorimeter (MicroCal) containing 20 µM Bcl-XL{Delta}TM. The sequence of the synthetic peptide is NH2-GTMENLSRRLKVTGDLFDIMSGQTDVDHPCOOH. Thermodynamic constants were determined by least squares fitting using Origin version 7.0 software (Origen Laboratories). For all analyses, n was forced to equal 1, and the initial ligand concentration (Bcl-2BD) was varied until a best fit was achieved. This operation was necessary, because the initial concentration of Bcl-2BD could not be determined spectroscopically due to a lack of UV light-absorbing amino acids (Trp or Tyr). Bcl-XL concentrations were determined by absorbance at 280 nm.


Figure 1
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  		FIGURE 1.
The BH3 domain of Beclin 1. A, domain organization of Beclin 1. Shown are Bcl-2 binding domain (residues 88-150) (Bcl-2BD), Bcl-2 homology domain 3 (residues 108-127) (BH3), and the evolutionarily conserved domain (residues 244-337) (ECD). B, aligned BH3 domains of human Bcl-2 family proteins. Circles above each column indicate residues buried (filled), partially buried (half-filled), or solvent-exposed (open) upon binding to Bcl-XL. Red arrows indicate critical helical contact residues that disrupt binding of Beclin 1 to Bcl-2/Bcl-XL.

 
Dynamic Light-scattering and Gel Filtration Analysis—Dynamic light-scattering data were collected on a Wyatt Technologies MiniDAWN instrument at 23 °C. All samples were diluted to 2 mg/ml in buffer A and passed through a 0.1-µm filter to remove particulate matter. Analytical gel filtration was performed using a Superdex-200 size exclusion column. For interaction assays between Bcl-XL{Delta}TM and Beclin Bcl-2BD, proteins were mixed at a 2:1 molar ratio, respectively, and injected onto a Superdex-200 size exclusion column equilibrated with buffer A. The elution profile of Bcl-XL{Delta}TM was monitored at 280 nm. In control experiments, the elution profile of wild-type and mutant Beclin Bcl-2BD proteins were determined by monitoring phenylalanine at 254 nm.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Beclin 1 Contains a Conserved BH3 Domain—Previous studies have identified amino acids 88-150 of Beclin 1 as the minimal Bcl-2 binding domain (Bcl-2BD) (22, 27). Bcl-2BD was shown to be necessary and sufficient for interaction with Bcl-2/Bcl-XL both in vivo and in vitro (22, 27, 28). The only surface on anti-apoptotic Bcl-2 family proteins known to mediate protein-protein interactions is a conserved hydrophobic groove responsible for binding the BH3 helix of BH3-only proteins (34-36). Therefore, we postulated that Beclin 1 may contain a BH3 domain. By performing primary sequence alignments between an array of BH3 domains from known Bcl-2 family members and Bcl-2BD, we were able to identify a putative BH3 domain near the N terminus of Beclin 1 just prior to the coiled coil domain (Fig. 1).

The Beclin 1 BH3 (BeclinBH3) domain shows a high degree of conservation at amino acids previously shown to be buried in the hydrophobic groove of Bcl-XL in the Bak (34), Bad (35), and Bim (36) peptide structures. These amino acids include Leu-112, Leu-116, Thr-119, Gly-120, and Phe-123 in human Beclin 1 (Fig. 1B). The BeclinBH3 domain also contains a signature glycine-aspartate dyad (Gly-120 and Asp-121), the aspartate of which forms a pair of conserved hydrogen bonds with Arg-139 lining the Bcl-XL groove.

Using a similar strategy, we searched for homology to other Bcl-2 homology domains (BH1, BH2, and BH3) but were unable to identify any significant similarity to such sequences. The presence of a BH3 domain, and only a BH3 domain, defines Beclin 1 as a novel member of the BH3-only family of proteins.

Mutations in BeclinBH3 Helical Contacts Disrupt Binding to Bcl-XL—To show that the putative BeclinBH3 is required for the interaction with pro-survival Bcl-2 family members, several conserved BH3 residues (Leu-116, Thr-119, and Gly-120) were individually mutated to glutamate, and the resulting mutant proteins were assayed for their ability to bind to Bcl-XL. Because of the poor solubility of full-length Beclin 1, Bcl-2BD was used for these experiments. Two assays, GST-mediated pulldown (Fig. 2A) and analytical gel filtration (Fig. 2B), consistently demonstrated that the missense mutations L116E or G120E led to abrogation of the interaction between Beclin 1 and Bcl-XL. A double mutant containing both the L116E and G120E mutations similarly lost binding to Bcl-XL. In contrast, the mutant T119E appeared to retain some binding to Bcl-XL in both assays.

Next, we determined the binding affinities using ITC, in which various Beclin 1 mutants (Bcl-2BD) were titrated into a sample chamber containing Bcl-XL protein. This analysis revealed an apparent binding affinity of 2.3 µM between wild-type Beclin 1 and Bcl-XL (Fig. 2C, left panel). A similar binding affinity, 1.1 µM, was attained using a synthetic BeclinBH3 peptide (Fig. 2D), suggesting that the extra sequences flanking the BH3 domain in Bcl-2BD do not make any significant contribution to Bcl-XL binding. Consistent with the pulldown and gel filtration assays, no interaction with Bcl-XL was observed for the mutant Beclin 1 Bcl-2BD-L116E (Fig. 2C, middle panel). Furthermore, in agreement with the observation that the Beclin 1 mutant T119E retained some binding to Bcl-XL in the pull-down and gel filtration assays (Fig. 2, A and B), T119E exhibited a decreased binding affinity (14.8 µM) for Bcl-XL by ITC (Fig. 2C, right panel). These results demonstrate that the BeclinBH3 domain itself is specifically required for the interaction between Beclin 1 and Bcl-XL in vitro. They also demonstrate that the BH3 domain of Beclin 1 can form a "stable" complex with Bcl-XL.


Figure 2
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  		FIGURE 2.
The BH3 domain of Beclin 1 is required for binding to Bcl-XL in vitro. A, GST-mediated pulldown assay using immobilized GST-Beclin-(88-156) (Bcl-2BD) and Bcl-XL{Delta}TM as the mobile phase. Shown are representative SDS-PAGE gels stained with Coomassie Blue. Two BeclinBH3 mutations, L116E and G120E, significantly abrogated binding of Bcl-XL, whereas a double mutant containing both mutations completely eliminated binding. The T119E mutant retains ~50% binding compared with wild type. Note that the intensity of the wild-type GST-fusion protein in the pulldown lane (lane 3, panel 2) is ~50% of the mutant fusions (lane 3, mutant panels). B, analytical gel filtration assay showing binding of GST-cleaved Bcl-2BD proteins to Bcl-XL. A 2-fold molar excess of Bcl-XL was mixed with wild-type or mutant Bcl2-BD and then injected onto a pre-equilibrated Superdex200 gel filtration column. The elution profile of Bcl-XL was monitored at 280 nm, which does not detect Bcl2-BD due to the lack of tryptophan and tyrosine. Only the wild type and the T119E mutant were able to cause a peak shift in Bcl-XL. Uncomplexed Bcl-XL (solid black line) was used as a control (gel not shown). Identical fractions were resolved by SDS-PAGE and visualized by Coomassie staining (right panel). C, binding isotherms generated using ITC. Binding affinities (Kd) were measured between the wild type (left), L116E (middle), and T119E (right) Bcl2-BD (ligand) and Bcl-XL (receptor). Consistent with the binding assays in A and B, the T119E mutant retained binding to Bcl-XL, whereas no significant heat release was observed for the L116E mutant. D, ITC of a 29-amino-acid synthetic BeclinBH3 peptide (ligand) and Bcl-XL (receptor).

 
A Beclin missense mutant, F123A in human Beclin 1, has recently been shown to disrupt binding to Bcl-2 in vivo (22). This mutation serendipitously maps to the C terminus of the BH3 helix in Beclin 1 (Fig. 1B). Yeast two-hybrid analysis and immunoprecipitation experiments in HEK293 cells suggest that this mutation disrupts the interaction between Beclin 1 and cellular Bcl-2 (22). Based on the Bim and Bad peptide structures, the benzene ring of this phenylalanine makes critical van der Waals contacts with amino acids in the conserved groove of Bcl-XL. These contacts are predicted to be significantly weakened or disrupted by mutation of Phe to the much smaller Ala. These results are consistent with our in vitro observations and suggest that the BeclinBH3 domain is responsible for interaction with Bcl-2 family proteins in vivo in the context of a fulllength protein. Thus, the BH3 domain of Beclin 1 is the minimal element necessary for interaction with anti-apoptotic Bcl-2 family proteins both in vitro and in vivo.

Overall Structure of the Bcl-XL-BeclinBH3 Heterodimer—BH3 domains may have been misidentified in a number of proteins (37) presumably because of the low complexity of the consensus sequence. This prompted us to examine the interaction between the BeclinBH3 domain and Bcl-XL in more detail. Accordingly, we co-crystallized a heterodimeric complex containing a BeclinBH3 peptide (hBeclin-(107-135)) and a deletion variant of Bcl-XL (Bcl-XL{Delta}loop{Delta}TM; see "Experimental Procedures"). The structure was determined at a 2.5 Å resolution by molecular replacement using the Bcl-XL-Bim structure (Table 1).


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  		TABLE 1
Statistics from crystallographic analysis

Rsym = {sum}h{sum}i |Ih,i - Ih|/{sum}h{sum}iIh,i, where Ih is the mean intensity of the i observations of symmetry-related reflections of h. R = {sum} |Fobs - Fcalc|/{sum}Fobs, where Fobs = Fp, and Fcalc is the calculated protein structure factor from the atomic model (Rfree was calculated with 5% of the reflections). Root mean square deviation in bond lengths and angles are the deviations from ideal values.

 
Unexpectedly, the heterodimer crystallized as a domainswapped tetramer, with each Bcl-XL monomer bound to an independent BeclinBH3 peptide and each Bcl-XL helix {alpha}1 intermolecularly "swapped" with its neighbor (Fig. 3A). Two lines of experimental evidence suggest that this domain swap was induced during crystallization, likely as a result of a crystallization artifact. First, dynamic light-scattering experiments revealed that the BeclinBH3-Bcl-XL complex reproducibly displayed a virtually identical hydrodynamic radius (Rh), 3.1 ± 0.2 nm, as unbound Bcl-XL in solution. Second, both the complex and unbound Bcl-XL showed identical elution volumes by analytical gel filtration, suggesting similar apparent molecular weights (data not shown). Bcl-XL{Delta}TM is monomeric in solution on the basis of reported NMR structure and analytical ultracentrifugation analysis (38, 39). Thus, by extension, the virtually identical solution properties we observed between the BeclinBH3 complex and Bcl-XL{Delta}TM indicates that the complex exists as a single heterodimer in solution, which only becomes domain-swapped during crystallization. It is important to note that the domain swap in Bcl-XL does not impact in any way the interactions between the BeclinBH3 peptide and Bcl-XL (Fig. 3A) and thus does not compromise the understanding of the molecular recognition between BeclinBH3 peptide and Bcl-XL.

Irrespective of the domain swapping, each dimeric unit aligns extremely well with the Bcl-XL-Bim structure, with a root mean square deviation of 0.531 Å over 584 backbone atoms (Fig. 3B). Helix {alpha}1 swapped into the exact position and orientation as it is normally found in the crystal structures of the BimBH3-Bcl-XL complex (root mean square deviation of 0.339 Å over 140 backbone atoms in helix {alpha}1) and Bcl-XL (root mean square deviation of 0.520 Å over 136 atoms in helix {alpha}1). This suggests that the overall fold, and more importantly the BH3 binding groove, remained intact during crystallization. Therefore, the interaction between the Beclin BH3 helix and Bcl-XL likely reflects their normal mode of interaction, unaffected by the domain swapping occurring on the opposite face of the molecule.

Overall, the BeclinBH3-Bcl-XL structure is similar to previously determined Bak (34), Bad (35), and Bim (36) peptide complexes. Each heterodimeric unit, composed of most of Bcl-XL chain A (residues 26-198), one BeclinBH3 peptide, and the first helix of Bcl-XL chain B (residues 2-25) forms a compact, globular fold. This fold has a core helix ({alpha}5) surrounded by seven shell helices ({alpha}1-{alpha}3, {alpha}4, and {alpha}6-{alpha}8) and intervening loops. A hydrophobic groove created by {alpha}2-{alpha}5 provides the docking surface for the BH3 peptide (Fig. 3C). The peptide adopts a single, six-turn, {alpha}-helix with BH3 residues Leu-112, Leu-116, Thr-119, Gly-120, Phe-123, and the aliphatic side chains of Ser-113 and Arg-115 forming a network of van der Waals interactions with the Bcl-XL groove (Fig. 3C). Several flanking hydrogen bonds stabilize the interaction and may provide the necessary specificity to orient the helix (Fig. 3C).


Figure 3
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  		FIGURE 3.
Structure of the Bcl-XL-BeclinBH3 peptide complex. A, overall structure of the domain-swapped tetramer composed of two Bcl-XL-BeclinBH3 heterodimers. B, structural alignment of one Bcl-XL-BeclinBH3 heterodimer and the Bcl-XL-BimBH3 heterodimer. Bcl-XL helix 1 ({alpha}12) in the Beclin structure (left) swapped into the precise position and orientation as the Bcl-XL helix 1 ({alpha}1) in the Bim (middle) complex, suggesting that the domain swap did not affect the integrity of the BH3 binding groove (see "Results" for details). C, detailed interaction surfaces between the BeclinBH3 domain and Bcl-XL. The proteins have been separated into components to help visualize the crowded interface. BH3 residues are shown in yellow and Bcl-XL residues in green. Hydrogen bonds are shown as dashed red lines. All figures were made with PyMOL (61), except Fig. 4, which was made with MOLSCRIPT software (62).

 
Interface between BeclinBH3 and Bcl-XL—Although there have been three reported structures of the Bcl-XL-BH3 complexes (34-36), only one was determined by x-ray crystallography at high resolution (36), which involves the Bim BH3 region. Analysis of the interface between the BeclinBH3 helix and Bcl-XL revealed a number of interesting features that are distinct from those of the Bcl-XL-Bim recognition.


Figure 4
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  		FIGURE 4.
Structural comparison of the BeclinBH3-Bcl-XL and BimBH3-Bcl-XL complexes. A, interface between the BeclinBH3 peptide and Bcl-XL in stereo. Side chains from residues in the BeclinBH3 peptide and Bcl-XL are yellow and green, respectively. Hydrogen bonds are represented as red dashed lines. B, interface between the BimBH3 peptide and Bcl-XL in stereo. Side chains from residues in the Bim peptide are colored gold.

 
Four hydrophobic and two charged residues in the BeclinBH3 helix, Leu-112, Leu-116, Lys-117, Thr-119, Asp-121, and Phe-123, mediate important interactions with surrounding residues in Bcl-XL (Fig. 4A). The corresponding residues in BimBH3 are Ile-90, Leu-94, Arg-95, Ile-97, Gly-98, Asp-99, and Phe-101, respectively (Fig. 4B). The interactions mediated by the two charged residues, Lys-117 and Asp-121 in Beclin 1, are highly conserved. In both Beclin 1 and Bim, the positively charged Lys-117/Arg-95 donates a hydrogen bond to Glu-129 in Bcl-XL. This interaction is stabilized by a pair of intramolecular contacts between Glu-129 and Arg-132 (Fig. 4). The invariant Asp-121/Asp-99 in Beclin/Bim make a pair of charge-stabilized hydrogen bonds to Arg-139 of Bcl-XL. In contrast to the two charged residues, interactions mediated by the four hydrophobic residues in Beclin 1 exhibit distinct features compared with those in the Bcl-XL-Bim complex (Fig. 4). One major difference is that the {alpha}3 helix in the BeclinBH3-bound Bcl-XL appears to be more flexible, as judged by the high temperature factors (B-factors) and poor electron density. Consequently, Leu-112 in Beclin 1 makes van der Waals contacts to Tyr-101 (but not Leu-108) in Bcl-XL (Fig. 4A), whereas the corresponding residue Ile-90 in Bim interacts with Leu-108 (but not Tyr-101). Similarly, Leu-116 in Beclin 1 interacts with Tyr-101 and Leu-108 (but not Phe-105) in Bcl-XL, whereas Leu-94 in Bim makes contact to Phe-105 (but not Tyr-101 or Leu-108). Apparently, these interaction features determine the distinct binding specificity of Beclin 1 toward Bcl-XL and perhaps other Bcl-2 family proteins.

A comparison of the crystal structure of unbound Bcl-XL with the BeclinBH3-Bcl-XL complex reveals similar structural rearrangements as seen in the Bim complex. Upon binding of the BH3 ligand, the {alpha}4 helix of Bcl-XL shifts toward the peptide by ~4 Å, whereas {alpha}3 opens up 4-5 Å (Fig. 5). The increased mobility of helix {alpha}3 in the Beclin complex is likely the result of BH3 peptide binding, as {alpha}3 also shows generally higher B-factors than other helices in the Bim peptide structure (36) but has normal B-factors in all available apoBcl-XL structures. In addition to this increased mobility, the side chain of Tyr-101 on helix {alpha}2, which lines the binding groove, is flipped with respect to the Bim-Bcl-XL structure (Fig. 5). We note that the conformation of this residue is particularly diverse, as it varies among both apoBcl-XL (compare accessions 1RD2 and 1MAZ) and holoBcl-XL (Fig. 5). It is possible that the orientation of Tyr-101 serves as a readout of the strength and/or specificity of BH3 binding. This speculation and other potential implications of the Tyr-101 conformations remain to be experimentally investigated.


Figure 5
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  		FIGURE 5.
Structural rearrangements of Bcl-XL upon binding of the BeclinBH3 domain. Shown are aligned backbone atoms from the following structures: unbound Bcl-XL (white), Bim bound Bcl-XL (blue), and Beclin 1-bound Bcl-XL (magenta). Significant rearrangements of Bcl-XL helices 3 and 4 as well as the side chain of Tyr-101 are shown.

 
The structure, combined with our biochemical results, validates the BH3 domain of Beclin as both necessary and sufficient for interaction with anti-apoptotic members of the Bcl-2 family. Thus, Beclin 1 can be definitively classified as a new member of the BH3-only family of proteins.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have shown that Beclin 1 is a new member of the expanding family of BH3-only proteins. Beclin 1, originally identified as a novel Bcl-2-interacting protein (27), was the first functional mammalian autophagy gene identified (23) and resides at a human tumor susceptibility locus on chromosome 17, which is monoallelically deleted in large numbers of breast, prostate, and ovarian cancers (24). Enforced expression of Beclin 1 in MCF-7 breast carcinoma cells, which show decreased levels of Beclin protein, reduces their tumorigenicity (23), and heterozygous disruption of beclin 1 in mice leads to suppression of autophagy and concomitant tumor formation (25, 26). These results have fueled the notion of autophagy as a general tumor-suppressive mechanism.

Surprisingly little is known about what Beclin 1 actually does in cells. Other than its constituency in a complex with the class III phosphoinositol 3-kinase Vps34p (40, 41) and its aforementioned interaction with Bcl-2/XL, the molecular mechanisms by which Beclin 1 functions remain poorly understood. A lack of known protein motifs as well as an unclear role in autophagy has confounded analysis of Beclin 1 in mammalian systems. Only two domains have been described in Beclin 1 prior to the BH3 domain, a coiled coil domain (residues 140-268) and a C-terminal evolutionarily conserved domain (residues 244-337) (42). The coiled coil domain interacts with two previously characterized partners, nPIST (43) and UVRAG (44), whereas the evolutionarily conserved domain is thought to bind Vps34p (42).

Our structural and biochemical characterization of Beclin 1 shows that its BH3 domain is both necessary and sufficient for interaction with Bcl-XL and that the proteins can form a stable complex in vitro. We further report the molecular details dictating the interaction of the two proteins. The identification of the Beclin BH3 domain implies that Beclin may be able to regulate Bcl-2, at least in some circumstances, rather than or in addition to the recently reported ability of Bcl-2 to regulate Beclin 1 (22). The literature is replete with examples of BH3-only proteins, which serve as death ligands, engaging down-stream multidomain Bcl-2 proteins prior to release of cyto-chrome c. Thus, there is ample precedent indicating that Beclin 1, which has now been shown to contain a BH3 domain, may function in a similar capacity.

There is a possibility, although unsupported, that the BH3 domain of Beclin 1 mediates a novel function with respect to the Bcl-2 family. For example, it could serve as an adaptor for loading mitochondria into autophagosomes, which has been previously suggested (45-47). Additionally, characterization of the BH3-only E3 ubiquitin ligase Arf-BP1/MULE has demonstrated that the BH3 domain, in at least one case, can be used for substrate recognition (48). It is worth noting, however, that a canonical death ligand function for its BH3 helix was, as the authors noted, not explicitly ruled out by the ability of Arf-BP1/MULE to polyubiquitinate Mcl-1 (48). That is, Arf-BP1/MULE may counter Mcl-1-mediated inhibition of Bak in addition to recognizing it as a substrate for ubiquitination. Nevertheless, the clear majority (if not all) of BH3-only proteins identified to date, including Arf-BP1/MULE, function in a pro-apoptotic manner upstream of Bcl-2 and Bax-like proteins. Given this precedent, it is not unreasonable to posit that Beclin 1 similarly contains a pro-apoptotic function mediated by its BH3 domain.

An unidentified apoptotic function may partially explain the ability of Beclin to suppress tumors. Three of nine previously described BH3-only proteins, Bim (49, 50), Bad (51), and PUMA (52), have been shown to function as tumor suppressors in mouse models, and virtually every BH3-only protein has been found to be dysregulated or mutated in at least one human cancer (11). Similarly, Beclin 1 is a BH3-only protein that can function as a tumor suppressor in mice and is deleted in many human cancers. Thus, Beclin fits the pattern of a canonical BH3-only protein extremely well and therefore is predicted to display similar activities. The generation of BH3 mutant knock-in mice should help clarify whether the BH3 domain of Beclin 1 is involved in its tumor suppressor function.

Paradoxically, defects in apoptosis were not observed upon UV irradiation or serum withdrawal of beclin 1-null mouse embryonic stem cells, suggesting that beclin 1 is not involved in apoptotic cell death (26). This negative result has been interpreted to indicate a novel, non-apoptotic (i.e. autophagic) tumor suppressor function for Beclin 1 (14, 26, 53). However, we note that one might not expect to see defects in apoptosis induced by such stimuli unless the particular pathways mediating their specific effects were additionally disrupted in beclin 1-null mouse embryonic stem cells. That is, the p53 pathway inducing Noxa and PUMA and the non-apoptotic AIF pathway, known to mediate the effects of UV damage and serum withdrawal, respectively (26), would need to be damaged in beclin 1-null Beclin cells in order for apoptosis to be affected. It is likely that both of these pathways are intact in beclin 1-null embryonic stem cells, although no evidence for or against this hypothesis has been presented to date. Beclin 1 might mediate apoptosis of an uncharacterized death stimulus (for example, nutrient deprivation), which would be missed if not assayed for explicitly in such experiments. Re-examination of the p53 and AIF pathways in beclin 1-null cells should help resolve this issue.

Further complicating a putative pro-apoptotic role for Beclin 1, several recent studies have suggested that autophagy can protect mitochondria from apoptotic stimuli (54, 55). For example, overexpression of Beclin 1 in COS-7 cells yielded a slight resistance to apoptosis induced by staurosporine, which was eliminated upon treatment of the autophagy inhibitor 3-methyladenine (55). This result suggests that the autophagy function of Beclin 1 can be cytoprotective in certain situations. Thus, Beclin 1 appears to have the ability to modulate the opposing survival and death functions of both apoptosis and autophagy. Any model of how Beclin 1 works would have to account for all of these apparently incompatible activities.

Although autophagic cell death has yet to be clearly demonstrated in mammalian cells containing intact apoptotic machinery, its existence would clearly necessitate a high degree of coordination between the disparate death pathways. The molecular mechanisms involved in coordinating the survival and death functions of autophagy, and each in turn with apoptosis, remain enigmatic. Autophagy, even in survival mode, is likely to be highly coordinated with apoptosis in order for the cell to switch from scavenging to dismantling. One possibility is that, under homeostatic conditions, Bcl-2 family proteins would suppress both apoptosis and autophagy, accomplishing the latter through direct binding to Beclin 1, whereas excessive cytotoxic stress would trigger classical apoptosis via BH3-only proteins (including Beclin 1 itself) but would default to autophagic cell death upon obstruction of apoptosis, such as through the lack of ATP or at a pathway lesion. Such a sequence of events would be consistent with recent insights suggesting that the disparate cell death mechanisms exist as a highly interwoven continuum of cellular pathways, which facilitates physiologic and pathologic responses to various stimuli (56, 57).

A significant question arising from this work is whether autophagy, in general, is tumor-suppressive or beclin 1 is a "special" autophagy gene with a secondary function in tumor suppression. It has been noted that oncogenes (e.g. phosphoinositol 3-kinase, Akt, Bcl-2) generally inhibit autophagy and that tumor suppressors (e.g. PTEN, p53, Beclin) generally activate autophagy (14, 29, 58). Nevertheless, a direct correlation between loss of autophagy and tumorigenesis has yet to be observed, as homozygous deletion of essential autophagy genes leads to early post-natal lethality in mice (59, 60). Thus, it remains a possibility that Beclin 1 is unique among autophagy genes and suppresses tumors through a previously unidentified function, separate from its ability to influence autophagy. Such a function might be provided by the BH3 domain.

In summary, we have shown that Beclin 1 is a novel BH3-only protein, which suggests a novel function upstream of Bcl-2/Bcl-XL. Further experiments will be required to determine the precise role of the Beclin BH3 domain in physiologic settings. Nonetheless, available observations suggest that Beclin 1 may contain a proapoptotic function, which could be related to its ability to suppress tumors in mammalian systems. A greater understanding of this mechanism will hopefully lead to new insights into the role of autophagy in cancer, as well as other autophagy-related diseases.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 2P1L) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported by Princeton University and grants from the National Institutes of Health. 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. Back

1 To whom correspondence should be addressed. Tel.: 609-258-6071; Fax: 609-258-6730; E-mail: ygshi{at}princeton.edu.

2 The abbreviations used are: BH, Bcl-2 homology; Bcl-2BD, Beclin 1 Bcl-2 binding domain; ITC, isothermal titration calorimetry; GST, glutathione S-transferase; Rh, hydrodynamic radius; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.
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