Crystal structure and biochemical analyses reveal Beclin 1 as a novel membrane binding protein - PubMed (original) (raw)

doi: 10.1038/cr.2012.24. Epub 2012 Feb 7.

Wooyoung Choi, Wanqiu Hu, Na Mi, Qiang Guo, Meisheng Ma, Mei Liu, Yuan Tian, Peilong Lu, Feng-Liang Wang, Haiteng Deng, Lei Liu, Ning Gao, Li Yu, Yigong Shi

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Crystal structure and biochemical analyses reveal Beclin 1 as a novel membrane binding protein

Weijiao Huang et al. Cell Res. 2012 Mar.

Abstract

The Beclin 1 gene is a haplo-insufficient tumor suppressor and plays an essential role in autophagy. However, the molecular mechanism by which Beclin 1 functions remains largely unknown. Here we report the crystal structure of the evolutionarily conserved domain (ECD) of Beclin 1 at 1.6 Å resolution. Beclin 1 ECD exhibits a previously unreported fold, with three structural repeats arranged symmetrically around a central axis. Beclin 1 ECD defines a novel class of membrane-binding domain, with a strong preference for lipid membrane enriched with cardiolipin. The tip of a surface loop in Beclin 1 ECD, comprising three aromatic amino acids, acts as a hydrophobic finger to associate with lipid membrane, consequently resulting in the deformation of membrane and liposomes. Mutation of these aromatic residues rendered Beclin 1 unable to stably associate with lipid membrane in vitro and unable to fully rescue autophagy in Beclin 1-knockdown cells in vivo. These observations form an important framework for deciphering the biological functions of Beclin 1.

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Figures

Figure 1

Figure 1

Structure of the Beclin 1 ECD. (A) Beclin 1 contains a BH3 domain, a coiled coil and an ECD. Rainbow color for the ECD domain is preserved in panel B. (B) Overall structure of the ECD (residues 248-447) of human Beclin 1. The structure is shown in rainbow color, with the N- and C-termini colored blue and red, respectively. An aromatic finger, comprising Phe359, Phe360 and Trp361, protrudes from the core structure. Beclin 1 ECD contains three structural repeats, each comprising a pair of β-strands and a long α-helix (top right). A deep pocket is located next to the aromatic finger (bottom right). (C) Sequence alignment of Beclin 1 homologs from multiple organisms. Conserved sequences are colored yellow. The three hydrophobic residues in the aromatic finger, highlighted in green, are highly conserved in all organisms except yeast. Secondary structural elements are indicated above the sequences. All structural images were generated using PyMol .

Figure 2

Figure 2

Beclin 1 ECD directly binds to lipid membrane. (A) Beclin 1 ECD associates with liposome by iodixanol gradient sedimentation analysis. Liposome co-sediments with low percentage of iodixanol, where Beclin 1 ECD was also detected (top panel). Free Beclin 1 ECD co-sediments with high percentage of iodixanol (top and middle panels). The control protein Nampt failed to co-sediment with liposome (bottom panel). (B) Beclin 1 ECD binds to liposome by ultracentrifugation analysis. Beclin 1 ECD or GST was ultracentrifuged in the presence or absence of liposome. The pellet and supernatant fractions were analyzed by SDS-PAGE and coomassie staining. (C) Analysis of liposome binding by Beclin 1 ECD. A fixed amount of liposome was ultracentrifuged in the presence of increasing amounts of Beclin 1 ECD. The pellets were analyzed by SDS-PAGE and coomassie staining. (D) Beclin 1 ECD preferentially binds to liposome enriched with cardiolipin. Liposomes of five distinct compositions were prepared, each containing fixed components (70% PC and 20% PE) and a variable component (10% each of the five specific phospholipids). Binding to Beclin 1 ECD was analyzed for these five liposomes. The control liposome has the same phospholipid composition as that of the Xenopus mitochondria .

Figure 3

Figure 3

Mutations in the aromatic finger of Beclin 1 ECD cripple its binding to mitochondria and liposomes. (A) The WT Beclin 1 ECD, but not the finger mutant (F359D/F360D/W361D), bound to liposome by ultracentrifugation analysis. (B) The WT Beclin 1 ECD, but not the finger mutant (F359D/F360D/W361D), exhibited a strong preference for cardiolipin-enriched liposomes. (C) A close-up view of the rationale for engineering a LC3 variant with grafted aromatic finger from Beclin 1. Amino acids 72-78 of LC3 constitute a surface loop with features similar to those of residues 354-363 of Beclin 1 ECD. Notably, the main chain Cα-Cα distances are similar in both cases. (D) The LC3 variant with engrafted aromatic finger, but not the WT LC3, associated with liposomes. S: supernatant; P: pellet (i.e., liposome fraction). (E) The LC3 variant with engrafted aromatic finger, but not the WT LC3, exhibited a strong preference for cardiolipin-enriched liposomes. (F) The full-length WT Beclin 1, but not the finger mutant (F359D/F360D/W361D), bound to liposome. S: supernatant; P: pellet (i.e., liposome fraction).

Figure 4

Figure 4

The Beclin 1 ECD deforms liposome. Free liposomes (panels A-C), liposomes pre-incubated with WT ECD (panels D-F), and liposomes pre-incubated with mutant ECD (panels G-I) were treated with nanogold particles and visualized under cryo-electron microscope. Beclin 1 ECD contains a 6×His tag, and the nanogold particle is linked to Ni2+-NTA. The WT Beclin 1 ECD, but not the mutant ECD, allowed the nanogold particles to be concentrated in the areas of liposome deformation, which might be liposome fusion, vesicle budding, and/or membrane vesiculation. (J) Quantitative analysis of liposome deformation by Beclin 1 ECD. The extent of liposome deformation is defined by the ratio of the longest dimension over the shortest dimension for each liposome. These ratios for 65 randomly chosen liposomes for each of three categories, control (no ECD), with WT ECD, and with mutant ECD, were measured and averaged. Liposomes incubated with WT ECD exhibited a larger average ratio than that for liposomes incubated with mutant ECD. **(K)**Incubation with Beclin 1 ECD led to increased radius for the liposomes. The increase is correlated with the concentrations of Beclin 1 ECD.

Figure 5

Figure 5

The aromatic finger of Beclin 1 plays an important role in autophagy.(A) Beclin 1-knockdown cells exhibited a markedly reduced level of autophagy upon starvation. Control and Beclin 1 stable knockdown NRK cells were stained with anti-LC3 antibody 4 h after starvation. Scale bar, 5 μm. (B) The WT Beclin 1, but not the finger mutant (F359D/F360D/W361D), rescued the autophagy defect in NRK cells. Beclin 1 stable knockdown NRK cells were transfected with WT or mutant Beclin 1-YFP. 24 h after transfection, cells were starved for 4 h. Then the cells were stained with anti-GFP or anti-LC3 antibody. Scale bar, 5 μm.(C) Average numbers of autophagosome per cell are shown for panels A and B. 50 cells were counted for each sample.(D) NRK cells transfected with the WT Beclin 1, but not with the finger mutant (F359D/F360D/W361D), had significant autophagic degradation of p62, a hallmark of autophagy. Cells from panels A and B were analyzed by western blot using antibodies specific for p62, Beclin 1 and actin. The position of transfected Beclin 1 is higher because Beclin 1 was fused to YFP.

Figure 6

Figure 6

The aromatic finger of Beclin 1 might be involved in omegasome formation.(A) The WT, full-length Beclin 1 exhibited punctate staining 30 min after starvation. Shown here are staining pattern for Beclin 1 in normal HeLa cells (WT), Beclin 1 stable knockdown HeLa cells (Beclin 1 KD), WT Beclin 1 stably expressing Beclin 1 KD cells (Beclin 1 KD+WT), mutant Beclin 1 stably expressing Beclin 1 KD cells (Beclin 1KD+MT). Scale bar, 10 μm. The right panel shows the expression levels of WT and mutant Beclin 1 in the 4 different types of cells. (B) The cellular localization of DFCP was restored by WT Beclin 1, but not mutant. Shown here are cellular staining pattern for DFCP in normal HeLa cells (WT), Beclin 1 KD cells, Beclin 1 KD+WT cells transfected with DFCP-GFP, and Beclin 1 KD+MT cells transfected with DFCP-GFP. 18 h after DFCP transfection, cells were starved for 0.5 h and stained for antibody against GFP. Scale bar, 5 μm. (C) The WT Beclin 1, but not the mutant, supported formation of a greater number of omegasomes (upper panel). Importantly, for the omegasome-containing cells, the average number of omegasomes in Beclin 1 KD+WT cells is ∼11.3, 57% more than that (7.2) in Beclin 1 KD+MT cells. Cells from panel B were quantified. 50 cells were counted for each category. Error bars represent standard deviation. For the top panel, the statistical P values are 0.0001/0.0055/0.0001 between “Cont” and “KD”/“KD+WT”/“KD+MT”, 0.0001/0.0001 between “KD” and “KD+WT”/“KD+MT”, and 0.0015 between “KD+WT” and “KD+MT”. For the bottom panel, the statistical P values are 0.0001/0.0022/0.0012 between “Cont” and “KD”/“KD+WT”/“KD+MT”, 0.0001/0.0216 between “KD” and “KD+WT”/“KD+MT”, and 0.0163 between “KD+WT” and “KD+MT”.

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