TY - JOUR
T1 - Structural adaptation of the glycophorin A transmembrane homodimer to D-amino acid modifications
AU - Gerber, Doron
AU - Sal-Man, Neta
AU - Shai, Yechiel
N1 - Funding 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.
PY - 2004/5/21
Y1 - 2004/5/21
N2 - 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.
AB - 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.
KW - D-amino peptide acids
KW - GPA, glycophorin A
KW - RP-HPLC, reverse phase high-performance liquid chromatography
KW - TFA, trifluoroacetic acid
KW - glycophorin A dimmer
KW - helix-helix interaction
KW - peptide recognition
KW - stereospecificity in assembly
UR - http://www.scopus.com/inward/record.url?scp=2342477732&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2004.03.004
DO - 10.1016/j.jmb.2004.03.004
M3 - Article
AN - SCOPUS:2342477732
SN - 0022-2836
VL - 339
SP - 243
EP - 250
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 1
ER -