title: 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-xL. 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 self-association 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 Ala176 and Gly180 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 His173 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.
authors.affiliation: Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005, USA.
authors.forename: Endah S
authors.initials: ES
authors.lastname: Sulistijo
authors.forename: Todd M
authors.initials: TM
authors.lastname: Jaszewski
authors.forename: Kevin R
authors.initials: KR
authors.lastname: MacKenzie
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane; D003902:Detergents; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008565:Membrane Proteins / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry*; D055672:Static Electricity; D025521:Tumor Suppressor Proteins
chemical_list: C090121:BNIP3 protein, human; C116838:BNIP3L protein, human; C474007:Bax protein (53-86); D003902:Detergents; D008565:Membrane Proteins; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D025521:Tumor Suppressor Proteins