Sequence-specific Dimerization of the Transmembrane Domain of the “BH3-only” Protein BNIP3 in Membranes and Detergent

Abstract

Mitochondria-mediated apoptosis is regulated by proteins of the Bcl-2 superfamily, most of which contain a C-terminal hydrophobic domain that plays a role in membrane targeting. Experiments with BNIP3 have implicated the transmembrane (TM) domain in its proapoptotic function, homodimerization, and interactions with Bcl-2 and Bcl-x L . We show that the BNIP3 TM domain self-associates strongly in Escherichia coli cell membranes and causes reversible dimerization of a soluble protein in the detergent SDS when expressed as an in-frame fusion. Limited mutational analysis identifies specific residues that are critical for BNIP3 TM selfassociation in membranes, and these residues are also important for dimerization in SDS micelles, suggesting that the self-association observed in membranes is preserved in detergent. The effects of sequence changes at positions Ala 176 and Gly 180 suggest that the BNIP3 TM domain associates using a variant of the GXXXG motif previously shown to be important in the dimerization of glycophorin A. The importance of residue His 173 in BNIP3 TM domain dimerization indicates that polar residues, which have been implicated in self-association of model TM peptides, can act in concert with the AXXXG motif to stabilize TM domain interactions. Our results demonstrate that the hydrophobic C-terminal TM domain of the pro-apoptotic BNIP3 protein dimerizes tightly in lipidic environments, and that this association has a strong sequence dependence but is independent of the identity of flanking regions. Thus, the transmembrane domain represents another region of the Bcl-2 superfamily of proteins that is capable of mediating strong and specific protein-protein interactions.The Bcl-2 superfamily of proteins plays a central role in regulating mitochondria-mediated apoptosis; the Bax subfamily promotes apoptosis, whereas the Bcl-2 subfamily protects against apoptosis (reviewed in Ref. 1). Protein-protein interactions between Bax and Bcl-2 subfamily members help determine cell fate (2, 3) and have accordingly been the subject of intensive study. Four regions of sequence homology, the BH1 through BH4 domains, contribute to the structure and function of these proteins, and the BH3 domain is particularly implicated in heterodimerization events (4 -9). Peptide and small molecule inhibitors of these protein-protein interactions can modulate apoptosis, further demonstrating the functional and pharmaceutical importance of these contacts (10 -13).Differentiation and development in metazoans requires celland signal-specific inputs to a functional Bcl-2/Bax checkpoint. The pro-apoptotic "BH3-only" proteins, which show homology to the Bcl-2 superfamily through the BH3 domain alone (14), are expressed or activated in specific tissues and in response to certain stimuli, making them candidates for connecting diverse signaling pathways to the ubiquitous apoptosis effector machinery (15, 16). BNIP3 (Bcl-2/19-kDa interacting protein 3) is a BH3-only protein whose expression and pro-apoptotic activity is induced following hyp...

Bnip3 Mediates the Hypoxia-induced Inhibition on Mammalian Target of Rapamycin by Interacting with Rheb

“…1c). Overexpressed Bnip3 has been shown to form a covalent dimer in a TM domain-dependent manner (58). Consistent with published data, the Bnip3-wt, Bnip3⌬N30, Bnip3⌬CD, Bnip3⌬BH3, and Bnip3⌬N114 showed up at twice the size of their predicted monomer molecular weights on the SDS-PAGE gel (Fig.…”

“…1c). In contrast, the Bnip3⌬TM could not form dimers and showed a molecular weight on SDS gel consistent with the predicted monomer (58,59). The mutant Rheb(C181S) protein migrated slower upon SDS-PAGE, which is consistent with earlier studies showing that isoprenylated Rheb migrates more quickly than the unmodified Rheb (60).…”

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

“…The membranes were formed from a 20-mg/ml solution of diphytanoyl-phosphatidylcholine (DPhPC; Avanti Polar Lipids) in decane. The measurements were performed at 23°C in 100-or 10-mM KCl solutions containing 1 mM EDTA and 13 C frequence of the C␤H3 group of Ala 176 from the three-dimensional 13 C F1-filtered/F3-edited-NOESY spectrum acquired for the "heterodimer" BNip3tm sample. The intermonomeric contacts are labeled.…”

“…As was demonstrated recently, the BNip3 TM segment exists in the form of a tightly associated dimer, in both the detergent and lipid environments (13). So far, the structure of the transmembrane domain of a representative of the Bcl-2 family in the lipid environment has not been reported.…”

The dimerization interface of the glycoprotein Ibβ transmembrane domain corresponds to polar residues within a leucine zipper motif

“…SDS-PAGE provides a simple, direct, and quick method to detect mutational effects on the TM-TM association. 10,17,21 As shown in Figure 4(A), the wild-type Ibb TM peptide (termed ''Ibb TMpep'') and three Ibb mutant TM peptides, mutation of Ala128 to Leu (IbbA128L TMpep), mutation of Gly136 to Leu (IbbG136L TMpep), and mutation of His139 to Leu (IbbH139L TMpep), were expressed in E. coli and purified by HPLC. Mutation of Gln129 to Leu (IbbQ129L TMpep) was not included Figure 3.…”