Structure of the IIA Domain of the Mannose Transporter from Escherichia coliat 1.7 Å Resolution

Abstract

The mannose transporter from Escherichia coliis a member of the phosphoenolpyruvate-dependent phosphotransferase system. The multisubunit complex couples translocation across the bacterial inner membrane with phosphorylation of the solute. A functional fragment (IIA Man, residues 2 to 133) of the membrane-associated IIAB Mansubunit of the mannose transporter was expressed as a selenomethionine protein, and the un- phosphorylated molecule was crystallized and its structure solved by X-ray crystallography. The protein consists of a central five-stranded β-sheet covered by helices on either face. The order of the secondary structure elements is (βα) 4,αβ. Four β-strands are arranged in a parallel manner with strand order 2134 and are linked by helices forming right-handed cross-over connections. The fifth strand that forms one edge of the sheet and runs antiparallel to the others is swapped between the subunits of the dimeric structure. Helices D and E form a helical hairpin. Histidine 10, which is transiently phosphorylated during catalysis, is located at the topologial switch-point of the structure, close to the subunit interface. Its imidazole ring is hydrogen bonded to the buried side-chain of Asp67. It is likely that Asp67 acts as a general base and thus increases the nucleophilicity of the histidine. Modeling suggests that the covalently bound phosphoryl group would be stabilized by the macrodipole of helix C. Putative interactions between IIA Manand the histidine-containing phosphocarrier protein are discussed.