STRUCTURAL COMPARISON OF PHOSPHORYLATED AND UNPHOSPHORYLATED FORMS OF III(GLC), A SIGNAL-TRANSDUCING PROTEIN FROM ESCHERICHIA-COLI, USING 3-DIMENSIONAL NMR TECHNIQUES

The 18.1-kDa protein III(Glc) from Escherichia coli acts as both a phosphocarrier protein in the phosphoenolpyruvate:glycose phosphotransferase system (PTS) and as a signal-transducing protein with respect to the uptake of non-PTS sugars. Phosphorylation of III(Glc) at the N(epsilon) (N3) position of His-90 was effected through a regeneration system that included MgCl2, DTT, excess PEP, and catalytic amounts of Enzyme I and HPr. NH, N-15, and C-13-alpha signal assignments for P-III(Glc) were made through comparison of N-15-H-1 correlation spectra (HSQC) of uniformly N-15-labeled preparations of phosphorylated and unphosphorylated protein and through analysis of three-dimensional triple-resonance HNCA spectra of P-III(Glc) uniformly labeled with both N-15 and C-13. Backbone and side-chain H-1 and C-13-beta signals were assigned using 3D heteronuclear HCCH-COSY and HCCH-TOCSY spectra of P-III(Glc). Using this approach, the assignments were made without reference to nuclear Overhauser effect data or assumptions regarding protein structure. The majority of NH, N-15, H-alpha, and C-13-alpha chemical shifts measured for P-III(Glc) were identical to those obtained for the unphosphorylated protein [Pelton, J. G., Torchia, D. A., Meadow, N. D., Wong C.-Y., & Roseman, S. (1991) Biochemistry 30, 100431. Those signals that exhibited shifts corresponded to residues within four segments (1) Leu-87-Gly-100, (2) Val-36-Val-46, (3) His-75-Ser-78, and (4) Ala-131-Val-138. These four segments are in close proximity to the active site residues His-75 and His-90 in the unphosphorylated protein [Worthylake, D., Meadow, N. D., Roseman, S., Liao, D., Hertzberg, O., & Remington, S. J. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 10382], and the chemical shift data provide strong evidence that if any structural changes accompany phosphorylation, they are confined to residues in these four segments. This conclusion is confirmed by comparing NOEs observed in 3D N-15/C-13 NOESY-HMQC spectra of the two forms of the protein. No NOE differences are seen for residues having the same chemical shifts in III(GlC) and P-III(Glc). Furthermore, with the exception of residues Ala-76, Asp-94, and Val-96, the NOEs of residues (in the four segments) which exhibited chemical shift differences also had the same NOEs in III(Glc) and P-III(Glc). In the case of residues Ala-76, Asp-94, and Val-96, minor differences in NOEs, corresponding to interproton distances changes of less than 1.5 angstrom, were observed. Together, the data show that no large structural rearrangements occur in III(Glc) upon phosphorylation and that the minor structural changes (< 1.5 angstrom) seen are limited to the active site. Hence, the results of structural studies of III(Glc) may be appropriate to modeling studies of the interaction of P-III(Glc) with HPr and other PTS and non-PTS proteins.