title: ATP-driven MalK dimer closure and reopening and conformational changes of the "EAA" motifs are crucial for function of the maltose ATP-binding cassette transporter (MalFGK2).
abstract: We have investigated conformational changes of the purified maltose ATP-binding cassette transporter (MalFGK(2)) upon binding of ATP. The transport complex is composed of a heterodimer of the hydrophobic subunits MalF and MalG constituting the translocation pore and of a homodimer of MalK, representing the ATP-hydrolyzing subunit. Substrate is delivered to the transporter in complex with periplasmic maltose-binding protein (MalE). Cross-linking experiments with a variant containing an A85C mutation within the Q-loop of each MalK monomer indicated an ATP-induced shortening of the distance between both monomers. Cross-linking caused a substantial inhibition of MalE-maltose-stimulated ATPase activity. We further demonstrated that a mutation affecting the "catalytic carboxylate" (E159Q) locks the MalK dimer in the closed state, whereas a transporter containing the "ABC signature" mutation Q140K permanently resides in the resting state. Cross-linking experiments with variants containing the A85C mutation combined with cysteine substitutions in the conserved EAA motifs of MalF and MalG, respectively, revealed close proximity of these residues in the resting state. The formation of a MalK-MalG heterodimer remained unchanged upon the addition of ATP, indicating that MalG-EAA moves along with MalK during dimer closure. In contrast, the yield of MalK-MalF dimers was substantially reduced. This might be taken as further evidence for asymmetric functions of both EAA motifs. Cross-linking also caused inhibition of ATPase activity, suggesting that transporter function requires conformational changes of both EAA motifs. Together, our data support ATP-driven MalK dimer closure and reopening as crucial steps in the translocation cycle of the intact maltose transporter and are discussed with respect to a current model.
authors.affiliation: Institut für Biologie/Bakterienphysiologie, Humboldt Universität zu Berlin, Chausseestrasse 117, D-10115 Berlin, Germany.
authors.forename: Martin L
authors.initials: ML
authors.lastname: Daus
authors.forename: Mathias
authors.initials: M
authors.lastname: Grote
authors.forename: Peter
authors.initials: P
authors.lastname: Müller
authors.forename: Meike
authors.initials: M
authors.lastname: Doebber
authors.forename: Andreas
authors.initials: A
authors.lastname: Herrmann
authors.forename: Heinz-Jürgen
authors.initials: HJ
authors.lastname: Steinhoff
authors.forename: Elie
authors.initials: E
authors.lastname: Dassa
authors.forename: Erwin
authors.initials: E
authors.lastname: Schneider
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000737:chemistry*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001692:Biological Transport; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000494:pharmacology; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology*; D008320:Maltose / Q000737:chemistry* / Q000378:metabolism; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000502:physiology*; D011510:Proteolipids / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; C461639:MalF protein, E coli; C079123:MalG protein, E coli; C489398:MalK protein, E coli; D009004:Monosaccharide Transport Proteins; D011510:Proteolipids; C461599:maltose transport system, E coli; C020485:proteoliposomes; D008320:Maltose; D000255:Adenosine Triphosphate