Unique Dimeric Structure of BNip3 Transmembrane Domain Suggests Membrane Permeabilization as a Cell Death Trigger

Abstract

BNip3 is a prominent representative of apoptotic Bcl-2 proteins with rather unique properties initiating an atypical programmed cell death pathway resembling both necrosis and apoptosis. Many Bcl-2 family proteins modulate the permeability state of the outer mitochondrial membrane by forming homoand hetero-oligomers. The structure and dynamics of the homodimeric transmembrane domain of BNip3 were investigated with the aid of solution NMR in lipid bicelles and molecular dynamics energy relaxation in an explicit lipid bilayer. The right-handed parallel helix-helix structure of the domain with a hydrogen bond-rich His-Ser node in the middle of the membrane, accessibility of the node for water, and continuous hydrophilic track across the membrane suggest that the domain can provide an ion-conducting pathway through the membrane. Incorporation of the BNip3 transmembrane domain into an artificial lipid bilayer resulted in pH-dependent conductivity increase. A possible biological implication of the findings in relation to triggering necrosis-like cell death by BNip3 is discussed.Mitochondria hold a crucial role in programmed cell death required to control cell development and to maintain homeostasis in multicellular organisms (1). Mitochondria-mediated cell death is both promoted and suppressed by apoptotic proteins of the Bcl-2 family, most of which contain a C-terminal hydrophobic domain essential for membrane targeting (2). A major function of Bcl-2 family proteins is to regulate the permeability state of the outer mitochondrial membrane by forming homo-and hetero-oligomers inside the membrane that determine cell fate (3-5). The pro-apoptotic protein BNip3 (Bcl-2 Nineteen-kDa interacting protein 3) with a single Bcl-2 homology 3 (BH3) domain is one of the most intensively studied members of the family (6). BNip3 and its homologues belong to an independent monophyletic branch with individual evolutionary history (2) and are essentially different from other BH3-only proteins such as Bid/Bik not only in that they do not require BH3 domain for their function but also because they directly cause changes of mitochondrial potential (7). BNip3-induced cell death is independent of caspases and cytochrome c release; it is believed to represent a novel form of programmed cell death, resembling necrosis rather than classical apoptosis (8).For all cells, loss of nutrient supply represents a potent signal for programmed death. BNip3 plays an important role in hypoxic cell death of normal and malignant cells (9). Hypoxia induces expression and accumulation of cytoplasmic or loosely membrane-bound BNip3; however, in order to activate cell death pathway acidosis is required (10). Transition from respiratory to glycolytic metabolism with increased glucose consumption, lactic acid production, and decrease of cytosolic pH causes redistribution of BNip3 to the outer mitochondrial membrane and integration of homodimeric BNip3 into it, triggering a cell death cascade, which ultimately leads to opening of the mitochondrial permeability tra...

A frequent, GxxxG-mediated, transmembrane association motif is optimized for the formation of interhelical Cα–H hydrogen bonds

“…As shown in Fig. 6A, the model is extremely similar to the NMR structure (16,20). The RMSD of the helical region of the entire TM domain is 1.10 ± 0.36 Å and only 0.56 ± 0.17 Å when it is computed only for the region that participates in the helix−helix interaction.…”

“…We tested CATM against five known homodimeric GAS right structures: glycophorin A (15), BNIP3 (16,20), and three members of the Tyrosine Receptor Kinase family, EphA1 (21), ErbB1 (EGFR) (22), and ErbB4 (23). We began testing using a simple combination of hydrogen bonding (E hb ) and van der Waals (E vdw ) to score the structural models.…”

The role of Bcl-2 family member BNIP3 in cell death and disease: NIPping at the heels of cell death

“…The unique structure of the TM domain suggests that BNIP3 dimers could act as proton channels in the outer mitochondrial membrane increasing ion conductance. 5 Serine 172 and histidine (His) 173 are residues that are present in the dimerization interface of BNIP3. These residues interact by hydrogen bonds and are essential for dimer formation.…”

Structure-Functional Insight Into Transmembrane Helix Dimerization

“…Correctness of the remaining models was assessed via comparison with the mutagenesis data. As a result, one of the final models consistent with the mutagenesis data was also in good agreement with the NMRderived structure of dimeric Bnip3 TM domain in lipid bicelles (Bocharov et al, 2007). Volynsky et al, 2010, used modeling methods in combination with ToxR assays to study dimerization of TM segments of ephrin receptor EphA1.…”

“…Investigation of spatial structure and internal dynamics of the homodimeric TM domain of human protein BNip3 (PDB 2J5D, Bocharov et al, 2007) revealed that in the lipid bicelles the central membrane-spanning -helices of BNip3 cross at the angle of -45° and form a right-handed parallel symmetric dimer via tetrad repeat pattern S 172 H 173 xxA 176 xxxG 180 xxxG 184 (Fig. 3).…”