Solution Structure of Enzyme IIA from the N,N -Diacetylchitobiose Branch of the Escherichia coli

The solution structure of trimeric Escherichia coli enzyme IIA (34 kDa), a component of the N,N -diacetylchitobiose/lactose branch of the phosphotransferase signal transduction system, has been determined by NMR spectroscopy. Backbone residual dipolar couplings were used to provide long range orientational restraints, and long range ( i j > 5 residues) nuclear Overhauser enhancement restraints were derived exclusively from samples in which at least one subunit was N/C/H/(Val-Leu-Ile)-methyl-protonated. Each subunit consists of a three-helix bundle. Hydrophobic residues lining helix 3 of each subunit are largely responsible for the formation of a parallel coiled-coil trimer. The active site histidines (His-89 from each subunit) are located in three symmetrically placed deep crevices located at the interface of two adjacent subunits (A and C, C and B, and B and A). Partially shielded from bulk solvent, structural modeling suggests that phosphorylated His-89 is stabilized by electrostatic interactions with the side chains of His-93 from the same subunit and Gln-91 from the adjacent subunit. Comparison with the x-ray structure of Lactobacillus lactis IIA reveals some substantial structural differences, particularly in regard to helix 3, which exhibits a 40° kink in IIA versus a 7° bend in IIA. This is associated with the presence of an unusually large (230-Å) buried hydrophobic cavity at the trimer interface in IIA that is reduced to only 45 Å in IIA.