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J Mol Biol. 2006 Dec 15;364(5):974-90. Epub 2006 Sep 29.

Sequence dependence of BNIP3 transmembrane domain dimerization implicates side-chain hydrogen bonding and a tandem GxxxG motif in specific helix-helix interactions.

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Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77005, USA.


The transmembrane domain of the pro-apoptotic protein BNIP3 self-associates strongly in membranes and in detergents. We have used site-directed mutagenesis to analyze the sequence dependence of BNIP3 transmembrane domain dimerization, from which we infer the physical basis for strong and specific helix-helix interactions in this system. Hydrophobic substitutions identify six residues as critical to dimerization, and the pattern of sensitive residues suggests that the BNIP3 helices interact at a right-handed crossing angle. Based on the dimerization propensities of single point mutants, we propose that: polar residues His173 and Ser172 make inter-monomer hydrogen bonds to one another through their side-chains; Ala176, Gly180, and Gly184 form a tandem GxxxG motif that allows close approach of the helices; and Ile183 makes inter-monomer van der Waals contacts. Since neither the tandem GxxxG motif nor the hydrogen bonding pair is sufficient to drive dimerization, our results demonstrate the importance of sequence context for either hydrogen bonding or GxxxG motif involvement in BNIP3 transmembrane helix-helix interactions. In this study, hydrophobic substitutions away from the six interfacial positions have almost no effect on dimerization, confirming the expectation that hydrophobic replacements affect helix-helix interactions only if they interfere with packing or hydrogen bonding by interfacial residues. However, changes to slightly polar residues are somewhat disruptive even when located away from the interface, and the degree of disruption correlates with the decrease in hydrophobicity. Changing the hydrophobicity of the BNIP3 transmembrane domain alters its helicity and protection of its backbone amides. We suggest that polar substitutions decrease the fraction of dimer by stabilizing an unfolded monomeric state of the transmembrane span, rather than by affecting helix-helix interactions. This result has broad implications for interpreting the sequence dependence of membrane protein stability in detergents.

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