Solution NMR Structure of the 48 kDa IIA-HPr Complex of the Escherichia coli Mannose Phosphotransferase System*

The solution structure of the 48 kDa IIAHPr complex of the mannose branch of the E. coli phosphotransferase system (PTS) has been solved by NMR using conjoined rigid body/torsion angle simulated annealing on the basis of intermolecular nuclear Overhauser enhancement data and residual dipolar couplings. IIA is dimeric and has two symmetrically related binding sites per dimer for HPr. A convex surface on HPr, formed primarily by helices 1 and 2, interacts with a deep groove at the interface of the two subunits of IIA. The interaction surface on IIA is predominantly helical, comprising helix 3 from the subunit that bears the active site His10, and helices 1, 4 and 5 from the other subunit. The total buried accessible surface area at the proteinprotein interface is 1450 Å. The binding sites on the two proteins are complementary in terms of shape, and distribution of hydrophobic, hydrophilic and charged residues. The active site histidines, His10 of IIA and His15 of HPr are in close proximity. An associative transition state involving a pentacoordinate phosphoryl group with trigonal bipyramidal geometry bonded to the N 2 atom of His10 and the N 1 atom of His15 can be readily formed with negligible displacement in the backbone coordinates of the residues immediately adjacent to the active site histidines. Comparison with the structures of complexes of HPr with three other structurally unrelated PTS proteins, enzyme I, IIA and IIA, reveals a number of common features that provide a molecular basis for understanding how HPr specifically recognizes a wide range of diverse proteins. The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) provides a signaling network whereby free energy generated from phosphoryl transfer between successive members of the pathway is transduced to drive the uptake of carbohydrates across the cytoplasmic membrane (1-4). The initial phosphorylation steps from phosphoenolpyruvate to enzyme I (EI) and subsequently to the histidine phosphocarrier protein, HPr, are common to all branches of the pathway. The subsequent phosphorylation steps involve sugar specific enzymes II which fall into four major families (glucose, mannitol, mannose and lactose/chitobiose) which bear no sequence and in the majority of cases no structural similarity to one another (2-4). The functional organization, however, of the enzymes II is similar: there are two cytoplasmic domains, IIA and IIB, and a transmembrane sugar permease domain, IIC (and in some instances IID as well), which may or may not be covalently linked to one another. The proteinprotein complexes within the PTS are generally weak and have proved refractory to crystallization. These complexes, in which proteins are able to recognize multiple and structurally diverse partners using similar interaction surfaces, provide a paradigm for understanding the structural basis of protein-protein interactions. In a series of papers, we have reported the solution structures of the enzyme I (N-terminal domain)-HPr complex (5), the two soluble complexes of the glucose branch of the PTS, IIA-HPr (6) and IIA-IIB (7), and the IIAHPr complex of the mannitol branch (8). In the present paper, we extend these studies to the solution structure determination of the 48 kDa dimeric IIA-HPr complex from the mannose branch of the PTS. The mannose transporter (II) is composed of four domains expressed as two proteins: the soluble IIAB component that associates with the integral membrane IICD permease (9, 10). The A and B domains of IIAB are linked by a 20-residue flexible linker and contain the first (His10) and JBC Papers in Press. Published on March 23, 2005 as Manuscript M501986200