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J. Biol. Chem., Vol. 281, Issue 7, 3856-3865, February 17, 2006

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ATP Induces Conformational Changes of Periplasmic Loop Regions of the Maltose ATP-binding Cassette Transporter^*

Martin L. Daus¹, Heidi Landmesser¹, Andreas Schlosser, Peter Müller^¶, Andreas Herrmann^¶, and Erwin Schneider²

From the Institut für Biologie/Bakterienphysiologie, Humboldt Universität zu Berlin, Chausseestrasse 117, D-10115 Berlin, Germany, the Institut für Medizinische Immunologie, Charité-Universitätsmedizin Berlin, Hessische Strasse 3-4, D-10115 Berlin, Germany, and the ^¶Institut für Biologie/Biophysik, Humboldt Universität zu Berlin, Invalidenstrasse 42, D-10115 Berlin, Germany

We have studied cofactor-induced conformational changes of the maltose ATP-binding cassette transporter by employing limited proteolysis in detergent solution. The transport complex consists of one copy each of the transmembrane subunits, MalF and MalG, and of two copies of the nucleotide-binding subunit, MalK. Transport activity further requires the periplasmic maltose-binding protein, MalE. Binding of ATP to the MalK subunits increased the susceptibility of two tryptic cleavage sites in the periplasmic loops P2 of MalF and P1 of MalG, respectively. Lys²⁶² of MalF and Arg⁷³ of MalG were identified as probable cleavage sites, resulting in two N-terminal peptide fragments of 29 and 8 kDa, respectively. Trapping the complex in the transition state by vanadate further stabilized the fragments. In contrast, the tryptic cleavage profile of MalK remained largely unchanged. ATP-induced conformational changes of MalF-P2 and MalG-P1 were supported by fluorescence spectroscopy of complex variants labeled with 2-(4'-maleimidoanilino)naphthalene-6-sulfonic acid. Limited proteolysis was subsequently used as a tool to study the consequences of mutations on the transport cycle. The results suggest that complex variants exhibiting a binding protein-independent phenotype (MalF500) or containing a mutation that affects the "catalytic carboxylate" (MalKE159Q) reside in a transition state-like conformation. A similar conclusion was drawn for a complex containing a replacement of MalKQ140 in the signature sequence by leucine, whereas substitution of lysine for Gln¹⁴⁰ appears to lock the transport complex in the ground state. Together, our data provide the first evidence for conformational changes of the transmembrane subunits of an ATP-binding cassette import system upon binding of ATP.

Received for publication, November 7, 2005 , and in revised form, December 7, 2005.

^* This work was supported by Grant SCHN 274/9-1 from the Deutsche Forschungsgemeinschaft. 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 These authors contributed equally to this work.

² To whom correspondence should be addressed: Institut für Biologie/Bakterienphysiologie, Humboldt Universität zu Berlin, Chausseestr. 117, D-10115 Berlin, Germany. Tel.: 49-030-2093-8121; Fax: 49-030-2093-8126; E-mail: erwin.schneider{at}rz.hu-berlin.de.

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