Intersubunit crosslinking of the heterotetrameric proton-translocating pyridine nucleotide transhydrogenase of Escherichia coli defines intersubunit contacts between transmembrane helices of the beta subunits.
The proton-translocating pyridine nucleotide transhydrogenase of Escherichia coli is composed of two types of subunits, alpha and beta, organized as an alpha(2)beta(2) tetramer. The protein contains three recognizable domains, of which domain II is the transmembrane region of the molecule containing the pathway for proton translocation. Domain II is composed of four transmembrane helices at the carboxyl-terminus of the alpha subunit and nine transmembrane helices at the amino-terminal region of the beta subunit. We have introduced pairs of cysteine residues into all of the loops connecting the transmembrane helices of domain II of the beta subunit. Crosslinking between the two beta subunits of the tetramer was induced spontaneously, or by treatment with cupric 1,10-phenanthrolinate or o-phenylenedimaleimide. Crosslinks between pairs of betaA114C, betaS183C, and betaA262C residues were observed, suggesting that pairs of domain II transmembrane helices 11, 12, and 14 were in proximity. These results, together with previous data (Bragg and Hou (2000) Biochem. Biophys. Res. Commun. 273, 955-959) suggest that the transhydrogenase tetramer is formed by apposition of alpha(2) and beta(2) dimers. Crosslinking between pairs of cysteine residues in the same beta subunit was not observed, possibly because the interhelical loops of the domain II region of the beta subunit were too short to allow correct orientation of the sulfhydryl groups for crosslinking.
Hou
C
C
Department of Biochemistry and Molecular Biology, University of British Columbia, 2146 Health Sciences Mall, Vancouver, British Columbia, V6T 1Z3.
Bragg
P D
PD
D000595:Amino Acid Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000378:metabolism; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D007425:Intracellular Membranes / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D009250:NADP Transhydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017830:Octoxynol; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D021122:Protein Subunits
D003432:Cross-Linking Reagents; D004220:Disulfides; D021122:Protein Subunits; D017830:Octoxynol; D009250:NADP Transhydrogenases; D003545:Cysteine