Structure of the soluble domain of a membrane-anchored thioredoxin-like protein from Bradyrhizobium japonicum reveals unusual properties

TlpA is an unusual thioredoxin-like protein present in the nitrogen-fixing soil bacterium Bradyrhizobium japonicum. A hydrophobic N-terminal transmemb rane domain anchors it to the cytoplasmic membrane, whereby the hydrophilic thioredoxin domain becomes exposed to the periplasmic space. There, TlpA c atalyses an essential reaction, probably a reduction,, in the biogenesis of cytochrome aa(3). The soluble thioredoxin domain (TlpA(sol)), devoid of th e membrane anchor, was purified and crystallized. Oxidized TlpA(sol) crysta llized as a non-covalent dimer in the space group P2(1)2(1)2(1). The X-ray structure analysis was carried out by isomorphous replacement using a xenon derivative. This resulted in a high-resolution (1.6 Angstrom) three-dimens ional structure that displayed all of the features of a classical thioredox in fold. A number of peculiar structural details were uncovered: (i) Only o ne of the two active-site-cysteine sulphurs (Cys72, the one closer to the N terminus) is exposed on the surface, making it the likely nucleophile for the reduction of target proteins. (ii) TlpA(sol) possesses a unique structu ral disulphide bond, formed between Cys10 and Cys155, which connects an unp recedented N-terminal a helix with a beta sheet near the C terminus. (iii) An insertion of about 25 an-Lino acid residues, not found in the thioredoxi n prototype of Escherichia coli, contributes only marginally to the thiored oxin fold, but forms an extra, surface-exposed a helix. This region plus an other surface-exposed stretch (-Ile-Gly-Arg-Ala-), which is absent even in the closest TlpA relatives, might be considered as specificity determinants for the recognition of target proteins in the periplasm. The TlpA(sol) str ucture paves the way towards unraveling important structure-function relati onships by rational mutagenesis. (C) 2001 Academic Press.