Intermonomer hydrogen bonds enhance gxxxg-driven dimerization of the BNIP3 transmembrane domain: Roles for sequence context in helix-helix association in membranes

Charles M. Lawrie, Endah S. Sulistijo, Kevin R. MacKenzie

Research output: Contribution to journalArticlepeer-review

22 Scopus citations

Abstract

We determined the sequence dependence of human BNIP3 transmembrane domain dimerization using the biological assay TOXCAT. Mutants in which intermonomer hydrogen bonds between Ser172 and His173 are abolished show moderate interaction, indicating that side-chain hydrogen bonds contribute to dimer stability but are not essential to dimerization. Mutants in which a GxxxG motif composed of Gly180 and Gly184 has been abolished show little or no interaction, demonstrating the critical nature of the GxxxG motif to BNIP3 dimerization. These findings show that side-chain hydrogen bonds can enhance the intrinsic dimerization of a GxxxG motif and that sequence context can control how hydrogen bonds influence helix-helix interactions in membranes. The dimer interface mapped by TOXCAT mutagenesis agrees closely with the interfaces observed in the NMR structure and inferred from mutational analysis of dimerization on SDS-PAGE, showing that the native dimer structure is retained in detergents. We show that TOXCAT and SDS-PAGE give complementary and consistent information about BNIP3 transmembrane domain dimerization: TOXCAT is insensitive to mutations that have modest effects on self-association in detergents but readily discriminates among mutations that completely disrupt detergent-resistant dimerization. The close agreement between conclusions reached from TOXCAT and SDS-PAGE data for BNIP3 suggests that accurate estimates of the relative effects of mutations on native-state protein-protein interactions can be obtained even when the detergent environment is strongly disruptive.

Original languageEnglish
Pages (from-to)924-936
Number of pages13
JournalJournal of Molecular Biology
Volume396
Issue number4
DOIs
StatePublished - 2010

Keywords

  • GxxxG motif
  • Hydrogen bonding
  • Membrane protein folding
  • Sequence context
  • Transmembrane domain dimerization

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