Solution structure of enzyme IIAChitobiose from the N,N′-diacetylchitobiose branch of the Escherichia coli phosphotransferase system

Abstract

The solution structure of trimeric Escherichia coli enzyme IIA Chb (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 15N/ 13C/2H/(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 Gin-91 from the adjacent subunit. Comparison with the x-ray structure of Lactobacillus lactis IIALac reveals some substantial structural differences, particularly in regard to helix 3, which exhibits a 40° kink in IIALac versus a 7° bend in IIAChb. This is associated with the presence of an unusually large (230- Å3) buried hydrophobic cavity at the trimer interface in IIALac that is reduced to only 45 Å3 in IIA Chb.