THE FRUCTOSE TRANSPORTER OF BACILLUS-SUBTILIS ENCODED BY THE LEV OPERON - BACKBONE ASSIGNMENT AND SECONDARY STRUCTURE OF THE IIBLEV SUBUNIT

The fructose transporter of the Bacillus subtilis phosphotransferase s ystem consists of two membrane associated (IIA and IIB) and two transm embrane (IIC and IID) subunits [Martin-Vestraete, I., Debarbouille, M. , Klier, A. & Rapoport, G. (1990) J. Mol. biol. 214, 657-671]. It medi ates uptake by a mechanism which couples translocation to phosphorylat ion of the transported solute. The 18-kDa IIBLev subunit transfers pho sphoryl groups from His9 of the IIA subunit to the sugar. The three-di mensional structure of IIBLev or similar proteins is not known. IIBLev was overexpressed in Escherichia coli and isotopically labelled with C-13/N-15 in H2O as well as in 70% D2O. N-15-edited NOESY, C-13-edited NOESY and C-13, N-15 triple-resonance experiments yielded a nearly co mplete assignment of the H-1, C-13 and N-15 resonances. Based on quali tative interpretation of NOE, scalar couplings, chemical shift values and amide exchange data, the secondary structure and topology of IIBLe v was determined. IIBLev comprises six parallel beta-strands, one anti parallel beta-strand and 5 alpha-helices. The order of the major secon dary-structure elements is (beta alpha)(5) beta (strand order 7651423) . Assuming that the beta alpha beta-motives form right-handed turn str uctures, helices alpha A and alpha B are packed to one face and helice s alpha C, alpha D and alpha E to the opposite face of the parallel be ta-sheet. His15 which is transiently phosphorylated during catalysis i s located in the loop beta 1/alpha A of the topological switch point. The amino terminal (beta/alpha)(4) part of IIBLev has the same topolog y as phosphoglyceromutase (PGM; PDB entry 3pgm). Both proteins catalyz e phosphoryltransfer reactions which proceed through phosphohistidine intermediates and they show a similar distribution of invariant residu es in the topologically equivalent positions of their active sites. Th e protein fold of IIBLev has no similarity to any of the known structu res of other yruvate-dependent-carbohydrate-phosphotransferase- system proteins.