Protein-protein recognition is an essential process in life. The chemistry of these kind of interactions is predominately stereospecific (i.e. receptor-ligand, antibody-hapten binding). Here, we investigated whether the hydrophobic nature of the membrane affects this stereospecificity. To this end, we synthesized a diastereomer analogue (2D-GPA) of the glycophorin A transmembrane helix, with two L-valine residues replaced by their D-enantiomer. This ensures a disruption of the secondary structure. We investigated the ability of the diastereomer peptide to recognize the GPA chimera in the ToxR homodimer reporting system, in vivo. The peptide demonstrated a dose-dependent dominant negative effect on the GPA transmembrane in the bacterial ToxR system, suggesting a wild-type like interaction. This result was corroborated in vitro by fluorescence energy transfer between 2D-GPA and all-L GPA. Peptide binding to the bacteria was confirmed through confocal imaging, and Western blot confirmed that ToxR GPA receptor levels are not affected by the presence of the exogenous peptide. In order to understand the structural basis for heterodimer formation, homodimer and heterodimer structures, based on the NMR 3D structure of GPA, were subjected to a molecular dynamics simulation. The resulting heterodimer structure maintained most of the original inter-helical interactions, and its structure is similar to that of the homodimer. We postulate that the need to satisfy all H-bonds can compensate for the structural strain induced by the presence of the D-amino acid residues.
|Number of pages||8|
|Journal||Journal of Molecular Biology|
|State||Published - 21 May 2004|
Bibliographical noteFunding Information:
We thank V. Kiss from the Weizmann Institute for his technical assistance with the fluorescence microscopy studies. This study was supported by the Joseph Cohen Minerva Center. The ToxR-GPA plasmid and the FHK12 E. coli strain were kindly supplied by D. Lamgosch. Y.S. holds the Harold S. and Harriet B. Brady Professional Chair in Cancer Research.
- D-amino peptide acids
- GPA, glycophorin A
- RP-HPLC, reverse phase high-performance liquid chromatography
- TFA, trifluoroacetic acid
- glycophorin A dimmer
- helix-helix interaction
- peptide recognition
- stereospecificity in assembly