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J. Biol. Chem., Vol. 281, Issue 18, 12833-12840, May 5, 2006

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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^*

Bettina Blüschke, Rudolf Volkmer-Engert, and Erwin Schneider¹

From the Institut für Biologie/Bakterienphysiologie, Humboldt Universität zu Berlin, Chausseestr. 117, D-10115 Berlin, Germany and the Institut für Medizinische Immunologie, Charité-Universitätsmedizin Berlin, Schumannstr. 20-21, D-10098 Berlin, Germany

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.

Received for publication, November 28, 2005 , and in revised form, February 23, 2006.

^* This work was supported by the Deutsche Forschungsgemeinschaft (SCHN 274/9-1). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 49-30-2093-8121; Fax: 49-30-2093-8126; E-mail: erwin.schneider{at}rz.hu-berlin.de.