THE STRUCTURE OF THE ESCHERICHIA-COLI PHOSPHOTRANSFERASE IIA(MANNITOL) REVEALS A NOVEL FOLD WITH 2 CONFORMATIONS OF THE ACTIVE-SITE

Background: The bacterial phosphoenolpyruvate-dependent phosphotransfe rase system (PTS) catalyses the cellular uptake and subsequent phospho rylation of carbohydrates. Moreover, the PTS plays a crucial role in t he global regulation of various metabolic pathways, The PTS consists o f two general proteins, enzyme I and the histidine-containing protein (HPr), and the carbohydrate-specific enzyme II [E-II]. E(II)s are usua lly composed of two cytoplasmic domains, IIA and IIB, and a transmembr ane domain, IIC. The IIA domains catalyse the transfer of a phosphoryl group from HPr to IIB, which phosphorylates the transported carbohydr ate. Knowledge of the structures of the IIA proteins may provide insig ht into the mechanisms by which the PTS couples phosphorylation reacti ons with carbohydrate specificity. Results: We have determined the cry stal structure of the Escherichia coli mannitol-specific IIA domain, I IA(mtl) (M-r 16.3 kDa), by multiple anomalous dispersion analysis of a selenomethionine variant of IIA(mtl). The structure was refined at 1. 8 Angstrom resolution to an R factor of 19.0% (R-free 24.2%). The enzy me consists of a single five-stranded mixed beta sheet, flanked by hel ices on both sides. The phosphorylation site (His65) is located at the end of the third beta strand, in a shallow crevice lined with hydroph obic residues. The sidechains of two conserved active-site residues, A rg49 and His111, adopt two different conformations in the four indepen dent IIA(mtl) molecules. Using a solution structure of phosphorylated HPr, and a combination of molecular modelling and NMR binding experime nts, structural models of the HPr-IIA(mtl) complex were generated. Con clusions: The fold of IIA(mtl) is completely different from the struct ures of other IIA proteins determined so far, The two conformations of Arg49 and His111 might represent different states of the active site, required for the different phosphoryl transfer reactions in which IIA (mtl) is involved. A comparison of the HPr-IIA(mtl) model with models of HPr in complex with other IIA enzymes shows that the overall intera ction mode between the two proteins is similar. Differences in the sta bilisation of the invariant residue Arg17 of HPr by the different IIA proteins might be part of a subtle mechanism to control the hierarchy of carbohydrate utilisation by the bacterium.