Solution Structure of the IIAChitobiose-IIBChitobiose Complex of the NN′-Diacetylchitobiose Branch of the Escherichia coli Phosphotransferase System

摘要

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 KD of ∼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 φ/ψ angles of Gly-13 from the right to left α-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.