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 (MalFGK(2))

Autor(en): Daus, Martin L.
Grote, Mathias
Mueller, Peter
Doebber, Meike
Herrmann, Andreas
Steinhoff, Heinz-Juergen 
Dassa, Elie
Schneider, Erwin 
Stichwörter: ABC TRANSPORTER; Biochemistry & Molecular Biology; CATALYTIC CYCLE; CROSS-LINKING; ESCHERICHIA-COLI; HUMAN P-GLYCOPROTEIN; MEMBRANE-PROTEIN; NUCLEOTIDE-BINDING; SIGNATURE MOTIFS; SUBUNIT INTERACTIONS; TRANSITION-STATE
Erscheinungsdatum: 2007
Herausgeber: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Journal: JOURNAL OF BIOLOGICAL CHEMISTRY
Volumen: 282
Ausgabe: 31
Startseite: 22387
Seitenende: 22396
Zusammenfassung: 
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.
ISSN: 00219258
DOI: 10.1074/jbc.M701979200

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