Specificity of membrane helix-helix interactions by mutagenesis and structural analysis

The activity of apoptosis protein BNIP3 has been associated with its ability to form homodimeric and heteromeric associations through its carboxy-terminal transmembrane domain (TMD), but little is known about the chemical or physical basis of these interactions. In this thesis, I describe two approaches to examine the sequence requirements for BNIP3 TMD dimerization and the properties that drive and stabilize this association. The first approach employs saturation mutagenesis to generate a library of single mutants in the context of a fusion protein construct and SDS-PAGE combined with Western blotting to characterize the mutant dimerization phenotypes. The mutagenesis data maps the BNIP3 TMD dimerization region and identifies five interacting residues for BNIP3 TMD dimerization: Ala176, Gly180, and Gly184 form tandem GxxxG motifs that allow close approach of the helix backbones, and His173 and Ser172 form inter-monomer hydrogen bonds. The mutagenesis data also show that the sequence context in which these five critical residues are embedded affects the strength of TMD helix-helix interactions because several mutations at or near the dimer interface that leave the small residues and the inter-monomer hydrogen bonding intact can abolish or profoundly lower dimerization. The second approach to the study of BNIP3 TMD dimerization involves determining the structure of the TMD dimer using solution NMR spectroscopy. Reconstitution of the BNIP3 TMD peptide in the non-ionic detergent dodecylphosphocholine (DPC) and addition of dipalmitoylphosphatidylcholine (DPPC) yields peptide spectra with excellent peak dispersion and resolution. This quality enables collection of chemical shift, J coupling, and NOE distance restraints, thus allowing determination of the BNIP3 TMD dimer structure. The NMR structure of the BNIP3 TMD dimer reveals the details of how the elements of the BNIP3 TMD sequence cooperate to support dimerization and provides a context to interpret the effects of the saturation mutagenesis results. Results from the saturation mutagenesis and structural analyses establish an understanding of the BNIP3 TMD dimerization and provide a framework for further studies of BNIP3, which include but are not limited to thermodynamic studies, functional analyses, and molecular dynamics modeling of the BNIP3 TMD associations