Topography of the surface of the signal-transducing protein EIIA(Glc) that interacts with the MalK subunits of the maltose ATP-binding cassette transporter (MalFGK2) of Salmonella typhimurium.
The signal-transducing protein EIIA(Glc), a component of the phosphoenolpyruvate-glucose phosphotransferase system, plays a key role in carbon regulation in enteric bacteria, such as Escherichia coli and Salmonella typhimurium. The phosphorylation state of EIIA(Glc) governs transport and metabolism of a number of carbohydrates. When glucose as preferred carbon source is transported, EIIA(Glc) becomes predominantly unphosphorylated and allosterically inhibits several permeases, including the maltose ATP-binding cassette transport system (MalFGK2) in a process termed "inducer exclusion." We have mapped the binding surface of EIIA(Glc) that interacts with the MalK subunits by using synthetic cellulose-bound peptide arrays like pep scan- and substitutional analyses. Three regions constituting two binding sites were identified encompassing residues 69-79 (I), 87-91 (II), and 118-127 (III). Region III is MalK-specific, whereas residues from regions I and II partly overlap but are not identical to the binding interfaces for interaction with glycerol kinase and lactose permease. These results were fully verified by studying the inhibitory effect of purified EIIA(Glc) variants carrying mutations at positions representative of each of the three regions on the ATPase activity of the purified maltose transport complex reconstituted into proteoliposomes. Moreover, a synthetic peptide encompassing residues 69-91 was demonstrated to partially inhibit ATPase activity. We also show for the first time that the N-terminal domain of EIIA(Glc) is essential for inducer exclusion.
Blüschke
Bettina
B
Institut für Biologie/Bakterienphysiologie, Humboldt Universität zu Berlin, Chausseestr. 117, D-10115 Berlin, Germany.
Volkmer-Engert
Rudolf
R
Schneider
Erwin
E
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry; D008320:Maltose / Q000737:chemistry; D008715:Methionine / Q000737:chemistry; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D012486:Salmonella typhimurium / Q000378:metabolism*; D013499:Surface Properties
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008081:Liposomes; C468279:MalFGK(2) protein, Salmonella typhimurium; C489399:MalK protein, Salmonella typhimurium; D009004:Monosaccharide Transport Proteins; C032242:crr protein, E coli; D008320:Maltose; D000255:Adenosine Triphosphate; D008715:Methionine; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System