Abstract
The solution structure of the IIA-IIB complex of the N,N'-diacetylchitobiose (Chb) transporter of the Escherichia coli phosphotransferase system has been solved by NMR. The active site His-89 of IIA(Chb) was mutated to Glu to mimic the phosphorylated state and the active site Cys-10 of IIB(Chb) was substituted by serine to prevent intermolecular disulfide bond formation. Binding is weak with a K(D) of approximately 1.3 mm. The two complementary interaction surfaces are largely hydrophobic, with the protruding active site loop (residues 9-16) of IIB(Chb) buried deep within the active site cleft formed at the interface of two adjacent subunits of the IIA(Chb) trimer. The central hydrophobic portion of the interface is surrounded by a ring of polar and charged residues that provide a relatively small number of electrostatic intermolecular interactions that serve to correctly align the two proteins. The conformation of the active site loop in unphosphorylated IIB(Chb) is inconsistent with the formation of a phosphoryl transition state intermediate because of steric hindrance, especially from the methyl group of Ala-12 of IIB(Chb). Phosphorylation of IIB(Chb) is accompanied by a conformational change within the active site loop such that its path from residues 11-13 follows a mirror-like image relative to that in the unphosphorylated state. This involves a transition of the phi/psi angles of Gly-13 from the right to left alpha-helical region, as well as smaller changes in the backbone torsion angles of Ala-12 and Met-14. The resulting active site conformation is fully compatible with the formation of the His-89-P-Cys-10 phosphoryl transition state without necessitating any change in relative translation or orientation of the two proteins within the complex.
Highlights
Ing a sequential series of bimolecular protein-protein complexes to active sugar translocation across the membrane and to regulation of an array of cellular processes, including carbon catabolite repression (1– 6)
We present the solution structure of the IIA-IIB complex of the Escherichia coli N,NЈ-diacetylchitobiose-specific enzyme II (IIChb), a representative of the lactose/ chitobiose branch of the phosphotransferase system (PTS) (38 – 41)
The active site residue, Cys-10, of IIBChb is located within an 8-residue protruding loop whose conformation is very similar to that of the low molecular weight protein-tyrosine phosphatases (42), including hydrogen-bonding interactions in the phosphorylated state between the phosphoryl group and backbone amide protons (11). (Note, throughout the text residues of IIBChb are shown in italics.)
Summary
Protein Expression and Mutagenesis—Genes encoding IIAChb* (corresponding to a N⌬13/D92L mutant of wild-type IIAChb) and IIBChb (kindly provided by Dr Saul Roseman, Johns Hopkins University, Baltimore) were cloned into the pET-11 vector. Additional H89E and C10S mutations of the active site residues of IIAChb* and IIBChb, respectively were introduced using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA). The H89E mutation in IIAChb* was designed to mimic the charge effects of phosphorylation of His-89, and the C10S mutation of IIBChb was introduced to prevent any potential complications arising from possible intermolecular disulfide bridge formation. The IIAChb*, IIAChb*(H89E), IIBChb, and IIBChb(C10S) plasmids were introduced into E. coli BL21(DE3) (Novagen) cells for protein expression and induced at an A600 ϳ0.8 with 1 mM isopropyl--D-thiogalactopyranoside at 37 °C. Labeling schemes for samples used for intermolecular NOE measurements on the IIAChb*(H89E)-IIBChb(C10S) complex
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