Phosphorylation of IIBChb 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 / angles of Gly-13 from the right to left ␣-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.The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) 3 couples a phosphorylation cascade involving 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). The first two steps of the PTS, involving autophosphorylation of enzyme I (EI) by phosphoenolpyruvate and subsequent phosphoryl transfer to the histidine phosphocarrier protein (HPr), are common to all branches of the pathway. The downstream components of the PTS comprise four major classes of sugar-specific enzymes II corresponding to the glucose (Glc), mannitol (Mtl), mannose (Man), and lactose/chitobiose (Chb) branches of the PTS. The enzymes II are generally organized into two cytoplasmic domains (IIA and IIB), and one transmembrane domain (IIC), which may or may not be covalently linked to one another. The phosphoryl group is transferred from HPr to IIA, from IIA to IIB and finally from IIB onto the incoming sugar bound to IIC. Despite their similar organization, the IIA and IIB domains of the different sugar-specific branches of the PTS bear no sequence similarity to one another and, with the exception of IIB Mtl (7,8) and IIB Chb (9 -11), no structural similarity either. Whereas structures of many of the individual cytoplasmic components of the PTS have been solved either by crystallography (9, 12-25) or NMR (7, 8, 10, 11, 26 -32), the complexes of the PTS have proved refractory to crystallization, presumably because of their weak and transient nature. Weak binding, however, is not an impediment to NMR spectroscopy, and over the last 10 years we have solved the solution structures of the N-terminal domain of enzyme I (EIN) complexed to HPr (33), and the IIA-HPr and IIA-IIB complexes of the glucose, mannitol, and mannose branches of the PTS (30, 31, 34 -37). These complexes provide a paradigm for understanding the structural basis of protein-protein interactions and how individual proteins can recognize multiple, structurally dissimilar, partners.In the present report, we present the solution structure of the IIA-IIB complex of the Escherichia coli N,NЈ-diacetylchitobiose-specific enzyme II (II Chb ), a representative of the lactose/ chitobiose branch of the PTS (38 -41). The A, B, and C domains of IIA Chb* , double mutant of IIA Chb comprising a 13-residue deletion at t...