Glucose transporter of Escherichia coli: NMR characterization of the phosphocysteine form of the IIBGlc domain and its binding interface with the IIA(Glc) subunit

The transmembrane subunit of the glucose transporter, IICBGlc, mediates vectorial transport with concomitant phosphorylation of glucose. Glucose 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-dimensional NMR experiments were used to characterize the phosphorylation of the 10 kDa subclonal IIB domain and the complementary binding interfaces of [N-15]IIB and [N-15]IIA(Glc). The largest chemical shift perturbations and the only NOE differences accompanying IIB phosphorylation are confined to the active site residue Cys35, as well as Ile36, Thr37, Arg38, Leu39, and Arg40, which are all located in the turn between strands beta 1 and beta 2 and on beta 2 itself. The significant increase of the amide cross-peak intensities of Ile36, Thr37, and Arg38 upon phosphorylation 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 experience chemical shift perturbations upon binding of IIA are located on a protruding surface formed by residues of strands beta 1, beta 2, and beta 4, the beta 4/alpha 3 loop, and residues from the first two turns of alpha 3. The corresponding binding surface of the IIA(Glc) domain is comprised of residues on five adjacent P-strands and two short helices surrounding the active site His90. The binding surface of IIA(Glc) for IIB coincides with the binding surface for HPr, the phosphoryl carrier 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].