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)

Martin L Daus,Mathias Grote,Peter Muöller,Meike Doebber,Andreas Herrmann,Heinz-Juörgen Steinhoff,Elie Dassa,Erwin Schneider

doi:10.1074/jbc.m701979200

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.

Highlights

ATP-binding cassette (ABC)3 transporters are involved in the uptake or export of an enormous variety of substances ranging from small ions to large polypeptides at the expense of ATP
The ABC domains are characterized by a set of Walker A and B motifs that are involved in nucleotide binding and by the unique “LSGGQ” signature sequence [3]
The transporter is composed of an extracellular receptor, the maltose-binding protein (MalE), and a membrane-bound complex (MalFGK2), comprising the pore-forming hydrophobic subunits, MalF and MalG, and two copies of the ABC subunit, MalK

Summary

Introduction

ATP-binding cassette (ABC) transporters are involved in the uptake or export of an enormous variety of substances ranging from small ions to large polypeptides at the expense of ATP. Genetic and biochemical evidence has given rise to the notion that ATP binding to the MalK dimer might be communicated via its Q-loops to the EAA motifs of MalFG [18, 19] In support of this view, the crystal structure of the vitamin B12 transporter of E. coli (BtuCD) revealed close contact between the Q-loop of BtuD and the EAA (or L) loops of BtuC and contacts of the Q-loop with atoms in the ATP binding site [13]. A molecular dynamics simulation suggests that conformational switching of the Q-loop may mediate communication between the TMDs and the catalytic sites [5]

Methods

Results

Conclusion