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

The solution structure of the 48-kDa IIAMan-HPr complex of the mannose branch of the Escherichia coli phosphotransferase system 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. IIAMan 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 IIAMan. The interaction surface on IIAMan is predominantly helical, comprising helix 3 from the subunit that bears the active site His-10 and helices 1, 4, and 5 from the other subunit. The total buried accessible surface area at the protein-protein interface is 1450 Å2. 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, His-10 of IIAMan and His-15 (italics indicate HPr residues) of HPr, are in close proximity. An associative transition state involving a pentacoordinate phosphoryl group with trigonal bipyramidal geometry bonded to the N-ϵ2 atom of His-10 and the N-δ1 atom of His-15 can be readily formed with negligible displacement in the backbone coordinates of the residues immediately adjacent to the active site histidines. Comparing the structures of complexes of HPr with three other structurally unrelated phosphotransferase system proteins, enzymes I, IIAglucose, and IIAmannitol, reveals a number of common features that provide a molecular basis for understanding how HPr specifically recognizes a wide range of diverse proteins.