GLUCOSE-TRANSPORTER OF ESCHERICHIA-COLI - NMR CHARACTERIZATION OF THEPHOSPHOCYSTEINE FORM OF THE IIBGLC DOMAIN AND ITS BINDING INTERFACE WITH THE IIA(GLC) SUBUNIT

The transmembrane subunit of the glucose transporter, IICBGlc, mediate s vectorial transport with concomitant phosphorylation of glucose. Glu cose phosphorylation proceeds through a cystein phosphate intermediate of the cytosolic IIB domain of IICGlc, which is phosphorylated by the IIA(Glc) subunit of the glucose transporter. Two- and three-dimension al NMR experiments were used to characterize the phosphorylation of th e 10 kDa subclonal IIB domain and the complementary binding interfaces of [N-15]IIB and [N-15]IIA(Glc). The largest chemical shift perturbat ions and the only NOE differences accompanying IIB phosphorylation are confined to the active site residue Cys35, as well as Ile36, Thr37, A rg38, Leu39, and Arg40, which are all located in the turn between stra nds beta 1 and beta 2 and on beta 2 itself. The significant increase o f the amide cross-peak intensities of Ile36, Thr37, and Arg38 upon pho sphorylation suggests that the conformational freedom of these groups becomes restrained, possibly due to hydrogen bonding to the oxygens of the bound phosphate and to interactions between the guanidinium group of Arg38 and the phosphoryl group. The residues of IIB which experien ce chemical shift perturbations upon binding of IIA are located on a p rotruding surface formed by residues of strands beta 1, beta 2, and be ta 4, the beta 4/alpha 3 loop, and residues from the first two turns o f alpha 3. The corresponding binding surface of the IIA(Glc) domain is comprised of residues on five adjacent P-strands and two short helice s surrounding the active site His90. The binding surface of IIA(Glc) f or IIB coincides with the binding surface for HPr, the phosphoryl carr ier protein by which IIA(Glc) is phosphorylated [Chen, Y., Reizer, J., Saier, M. H., Fairbrother, W. J., & Wright, P. E. (1993) Biochemistry 32, 32-37].