The Membrane Topology of Proton-pumping Escherichia coli Transhydrogenase Determined by Cysteine Labeling

Abstract

The membrane topology of proton-pumping nicotinamide-nucleotide transhydrogenase from Escherichia coli was determined by site-specific chemical labeling. A His-tagged cysteine-free transhydrogenase was used to introduce unique cysteines in positions corresponding to potential membrane loops. The cysteines were reacted with fluorescent reagents, fluorescein 5-maleimide or 2-[(4 -maleimidyl)anilino]naphthalene-6-sulfonic acid, in both intact cells and inside-out vesicles. Labeled transhydrogenase was purified with a small-scale procedure using a metal affinity resin, and the amount of labeling was measured as fluorescence on UV-illuminated acrylamide gels. The difference in labeling between intact cells and inside-out vesicles was used to discriminate between a periplasmic and a cytosolic location of the residues. The membrane region was found to be composed of 13 helices (four in the ␣-subunit and nine in the ␤-subunit), with the C terminus of the ␣-subunit and the N terminus of the ␤-subunit facing the cytosolic and periplasmic sides, respectively. These results differ from previous models with regard to both number of helices and the relative location and orientation of certain helices. This study constitutes the first in which all transmembrane segments of transhydrogenase have been experimentally determined and provides an explanation for the different topologies of the mitochondrial and E. coli transhydrogenases.Nicotinamide-nucleotide transhydrogenases are integral membrane proteins that catalyze the reduction of NADP ϩ by NADH with a concomitant translocation of protons across the membrane, e.g. from the periplasm or intermembrane space to the cytosol or matrix in bacteria and mitochondria, respectively, according to the following:Transhydrogenases are conformationally driven proton pumps in which binding and release of NADP(H) are thought to cause the structural changes allowing hydride transfer as well as proton translocation to occur (1). It is generally concluded that the H ϩ /H Ϫ ratio of the transhydrogenase reaction is 1 (2-4). At present, transhydrogenases from 12 different species have been cloned and/or sequenced that show an overall amino acid sequence identity of 23%. All transhydrogenases possess the same structural organization, regardless of primary sequence arrangement, with three major domains: two hydrophilic domains (domains I and III) comprising a 400-residue-long NAD(H)-binding domain and a 200-residue-long NADP(H)-binding domain, respectively, and domain II, containing a 360-residue-long hydrophobic domain that constitutes the membrane-spanning part of the enzyme (for reviews, see Refs. 4 and 5). The hydrophilic domains have been shown to protrude into the cytosol or matrix in bacteria and mitochondria, respectively (6, 7).Escherichia coli transhydrogenase consists of two subunits, ␣ (510 residues) and ␤ (464 residues), assembled as an ␣ 2 ␤ 2 -tetramer. The membrane domain is composed of the 110 Cterminal residues of the ␣-subunit and the 260 N-terminal residues of the ␤-subunit and ...

The Transmembrane Domains of the ABC Multidrug Transporter LmrA Form a Cytoplasmic Exposed, Aqueous Chamber within the Membrane

“…In previous studies, fluorescein maleimide, which at neutral pH is a dianion and membrane-impermeable, proved to be a useful fluorophore to determine the membrane topology of integral membrane proteins by modification of engineered cysteines (21)(22)(23). It was found that this thiol reagent does not react with cysteines in the lipidic membrane environment, but readily reacts with cysteines in polar loop regions.…”

“…Refs. [21][22][23]. Fluorescein maleimide is therefore a suitable probe to determine whether the cysteine is situated in a polar loop region or within a lipidic membrane region.…”

Review and Hypothesis. New insights into the reaction mechanism of transhydrogenase: Swivelling the dIII component may gate the proton channel

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