TY - JOUR
T1 - Hetero-assembly between all-L- and all-D-amino acid transmembrane domains
T2 - Forces involved and implication for inactivation of membrane proteins
AU - Sal-Man, Neta
AU - Gerber, Doron
AU - Shai, Yechiel
N1 - Funding Information:
This study was supported by the Josef Cohn Minerva Center. We would like to thank Dr D. Langosch from Heidelberg University, who provided the ToxR-GPA and A 16 plasmids as well as the FHK12 and PD28 E. coli strains. We thank V. Kiss from the Weizmann Institute for his technical assistance in the microscopy studies, Dr R. Barak for her technical assistance in the chemotaxis assay, and Dr M. Eisenbach and Dr A. H. Futerman for their comments and critical reading of the manuscript.
PY - 2004/11/26
Y1 - 2004/11/26
N2 - Protein-protein interactions within the membrane, partially or fully mediated by transmembrane (TM) domains, are involved in many vital cellular processes. Since the unique feature of the membrane environment enables protein-protein assembly that otherwise is not energetically favored in solution, the structural restrictions involved in the assembly of soluble proteins are not necessarily valid for the assembly of TM domains. Here we used the N-terminal TM domain (Tar-1) of the Escherichia coli aspartate receptor as a model system for examining the stereospecificity of TM-TM interactions in vitro and in vivo in isolated systems, and in the context of the full receptor. For this propose, we synthesized Tar-1 all-l and all-d amino acid TM peptides, a mutant TM peptide and an unrelated TM peptide. The data revealed: (i) Tar-1 all-d specifically associated with Tar-1 all-l within a model lipid membrane, as determined by using fluorescence energy transfer experiments; (ii) Tar-1 all-l and all-d, but not the control peptides, demonstrated a dose-dependant dominant negative effect on the Tar-1 TM homodimerization in the bacterial ToxR assembly system, suggesting a wild-type-like interaction; and most interestingly, (iii) both Tar-1 all-l and all-d showed a remarkable ability to inhibit the chemotaxis response of the full-length receptor, in vivo. Peptide binding to the bacteria was confirmed through confocal imaging, and Western blotting confirmed that ToxR Tar-1 chimera protein levels are not affected by the presence of the exogenous peptides. These findings present the first evidence that an all-d TM domain peptide acts in vivo similarly to its parental all-l peptide and suggest that the dimerization of the TM domains is mainly mediated by side-chain interactions, rather than geometrically fitted conformations. In addition, the study provides a new approach for modifying the function of membrane proteins by proteolysis-free peptides.
AB - Protein-protein interactions within the membrane, partially or fully mediated by transmembrane (TM) domains, are involved in many vital cellular processes. Since the unique feature of the membrane environment enables protein-protein assembly that otherwise is not energetically favored in solution, the structural restrictions involved in the assembly of soluble proteins are not necessarily valid for the assembly of TM domains. Here we used the N-terminal TM domain (Tar-1) of the Escherichia coli aspartate receptor as a model system for examining the stereospecificity of TM-TM interactions in vitro and in vivo in isolated systems, and in the context of the full receptor. For this propose, we synthesized Tar-1 all-l and all-d amino acid TM peptides, a mutant TM peptide and an unrelated TM peptide. The data revealed: (i) Tar-1 all-d specifically associated with Tar-1 all-l within a model lipid membrane, as determined by using fluorescence energy transfer experiments; (ii) Tar-1 all-l and all-d, but not the control peptides, demonstrated a dose-dependant dominant negative effect on the Tar-1 TM homodimerization in the bacterial ToxR assembly system, suggesting a wild-type-like interaction; and most interestingly, (iii) both Tar-1 all-l and all-d showed a remarkable ability to inhibit the chemotaxis response of the full-length receptor, in vivo. Peptide binding to the bacteria was confirmed through confocal imaging, and Western blotting confirmed that ToxR Tar-1 chimera protein levels are not affected by the presence of the exogenous peptides. These findings present the first evidence that an all-d TM domain peptide acts in vivo similarly to its parental all-l peptide and suggest that the dimerization of the TM domains is mainly mediated by side-chain interactions, rather than geometrically fitted conformations. In addition, the study provides a new approach for modifying the function of membrane proteins by proteolysis-free peptides.
KW - Tar receptor
KW - chirality
KW - dimerization
KW - peptide
KW - transmembrane domain
UR - http://www.scopus.com/inward/record.url?scp=7944224560&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2004.09.066
DO - 10.1016/j.jmb.2004.09.066
M3 - Article
AN - SCOPUS:7944224560
SN - 0022-2836
VL - 344
SP - 855
EP - 864
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 3
ER -