Crosslinking between α and β Subunits Defines the Orientation and Spatial Relationship of Some of the Transmembrane Helices of the Proton-Translocating Pyridine Nucleotide Transhydrogenase of Escherichia coli

• Published in: Biochemical and biophysical research communications 2000-07, Vol.273 (3), p.955-959
• Main Authors: Bragg, Philip D., Hou, Cynthia
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Electrophoresis, Polyacrylamide Gel; Escherichia coli - enzymology; Ion Transport; Molecular Sequence Data; Molecular Weight; NADP Transhydrogenases - chemistry; NADP Transhydrogenases - metabolism; Protons
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1006/bbrc.2000.3037

The proton-translocating pyridine nucleotide transhydrogenase of Escherichia coli is composed of two types of subunits, α and β, organized as an α2β2 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 α subunit and either eight or nine transmembrane helices at the amino-terminal region of the β subunit. We have introduced pairs of cysteine residues into a cysteine-free transhydrogenase by site-directed mutagenesis. Disulfide bond formation between some of these cysteine residues occurred spontaneously or on treatment with cupric 1,10-phenanthrolinate. Analysis of crosslinked products confirmed that there are nine transmembrane helices in the domain II region of the β subunit. The proximity to one another of several of the transmembrane helices was determined. Thus, helices 2 and 4 are close to helix 6 (nomenclature of Meuller and Rydström, J. Biol. Chem. 274, 19072–19080, 1999), and helix 3 and the carboxyl-terminal eight residues of the α subunit are close to helix 7. In the α2β2 tetramer, helices 2 and 4 of one α subunit are close to the same pair of transmembrane helices of the other α subunit, and helix 6 of one β subunit is close to helix 6 of the other β subunit.