Topography of the Surface of the Escherichia coli Phosphotransferase System Protein Enzyme IIA^glc that Interacts with Lactose Permease

Abstract

The unphosphorylated form of enzyme IIAglc of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system inhibits transport catalyzed by lactose permease. We (Seok et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 13515-13519) previously characterized the area on the cytoplasmic face of lactose permease that interacts with enzyme IIAglc, using radioactive enzyme IIAglc. Subsequent studies (Sondej et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 3525-3530) suggested consensus binding sequences on proteins that interact with enzyme IIAglc. The present study characterizes a region on the surface of enzyme IIAglc that interfaces with lactose permease. Acetylation of lysine residues by sulfosuccinimidyl acetate treatment of enzyme IIAglc, but not lactose permease, reduced the degree of interaction between the two proteins. To localize the lysine residue(s) on enzyme IIAglc that is(are) involved in the regulatory interaction, selected lysine residues were mutagenized. Conversion of nine separate lysines to glutamic acid resulted in proteins that were still capable of phosphoryl acceptance from HPr. Except for Lys69, all the modified proteins were as effective as the wild-type enzyme IIAglc in a test for binding to lactose permease. The Lys69 mutant was also defective in phosphoryl transfer to glucose permease. To derive further information concerning the contact surface, additional selected residues in the vicinity of Lys69 were mutagenized and tested for binding to lactose permease. On the basis of these studies, a model for the region of the surface of enzyme IIAglc that interacts with lactose permease is proposed.

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

“…Point mutations in several well-conserved residues of EIIA Glc also lower the phosphoryl transfer activity but only by between 20 and 70% (822). It was concluded from these observations (98) that multiple interactions contribute to the affinity between PTS protein partners.…”

“…Other observations support this interpretation. When galactosides are present at saturating amounts, 125 I-labeled unphosphorylated EIIA Glc binds in a 1:6 stoichiometry to wild-type LacY (822,987). Some of these residues are also important for the interaction of EIIA Glc with HPr, EIIB Glc , and GlpK (Table 2).…”

Topography of the Surface of the Signal-transducing Protein EIIAGlc That Interacts with the MalK Subunits of the Maltose ATP-binding Cassette Transporter (MalFGK2) of Salmonella typhimurium

“…None of the hydrophobic residues (underlined) can be replaced by amino acids with charged or polar side chains without loss of binding, suggesting that the binding interface is largely hydrophobic. Regions 1 and 2 basically encompass ␤-strands 5-7 of enzyme IIA Glc , which have also been implicated in recognizing other target proteins (4,5,(7)(8)(9) (Fig. 3).…”

“…Our results suggest that two binding interfaces exist; one partly overlaps but is not identical to that for interaction with glycerol kinase (4,5) and lactose permease (9), whereas the other is unique for MalK. (13), and MalE (18) of S. typhimurium were purified as described.…”

“…Structural analyses of complexes of EIIA Glc with glycerol kinase (5), glucose permease (EIICB) (6,7), and HPr (8) largely confirmed this notion. For lactose permease, a biochemical analysis also gave rise to a model that suggests an overlap of the interacting surface of EIIA Glc with that for interaction with other proteins (9). Thus far, nothing is known on the binding site by which EIIA Glc interacts with the maltose/maltodextrin ATP-binding cassette transport system.…”