On parallel and antiparallel topology of a homodimeric multidrug transporter.
The recently suggested antiparallel topology of EmrE has intriguing implications for many aspects of the biology of ion-coupled transporters. However, it is at odds with biochemical data that demonstrated the same topology for all protomers in the intact cell and with extensive cross-linking studies. To examine this apparent contradiction we chemically cross-linked dimers with a rigid bifunctional maleimide using Cys replacements at positions not permissible by an antiparallel topology. A purified cross-linked dimer binds substrate and transports it in proteoliposomes with kinetic constants similar to those of the non-cross-linked dimer. The cross-linked dimers do not interact with non-cross-linked dimers as judged from the fact that inactive mutants do not affect their activity (negative dominance). The results support the contention that EmrE with parallel topology is fully functional. We show that the detergents used in crystallization increase the fraction of monomers in solution. We suggest that the antiparallel orientation observed is a result of the arrangement of the monomers in the crystal. Functionality of EmrE with the suggested antiparallel orientation of the monomers remains to be characterized.
Soskine
Misha
M
Alexander A. Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
Mark
Shirley
S
Tayer
Naama
N
Mizrachi
Roy
R
Schuldiner
Shimon
S
D017920:Antiporters / Q000737:chemistry* / Q000502:physiology; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D003902:Detergents / Q000494:pharmacology; D019281:Dimerization; D018432:Drug Resistance, Multiple; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000502:physiology; D006358:Hot Temperature; D007700:Kinetics; D008301:Maleimides / Q000494:pharmacology; D008958:Models, Molecular; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010269:Paraquat / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation
D017920:Antiporters; D003432:Cross-Linking Reagents; D003545:Cysteine; D003902:Detergents; D029968:Escherichia coli Proteins; D008301:Maleimides; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D010269:Paraquat; C075436:EmrE protein, E coli; C043592:maleimide