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. 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. They are found in all organisms from bacteria to humans, and dysfunction is often associated with disease in humans, such as cystic fibrosis, adrenoleukodystrophy, or Stargardt's macular dystrophy (1). ABC transporters share a common architectural organization comprising two hydrophobic transmembrane domains (TMDs) that form the translocation pathway and two hydrophilic nucleotide binding (ABC) domains (NBDs) that hydrolyze ATP. In fact, in most prokaryote importers, TMDs and NBDs are expressed as separate protein subunits, whereas in most export systems of both prokaryotes and eukaryotes, they are usually fused into a single polypeptide chain (2).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 crystal structures of several mostly prokaryotic NBDs have been reported that largely agree on the overall fold (reviewed in Refs. 4 -6). Accordingly, the cassette can be divided into a RecA-like subdomain encompassing both Walker m...

PhoU Allows Rapid Adaptation to High Phosphate Concentrations by Modulating PstSCAB Transport Rate in Sinorhizobium meliloti

“…It has been suggested that PhoU modulates the kinase/phosphatase activity of PhoR in response to the conformational state of PstSCAB (5). Previous work with the E. coli MalFGK 2 maltose transporter indicated that Q140K and E158Q substitutions within the MalK ATP binding subunit locked the transporter in the open and closed states, respectively (39). Both of these residues are conserved in the PstB subunit of the S. meliloti PstSCAB transporter (Fig.…”

“…S7 in the supplemental material). Mutant pstB alleles were constructed to produce PstB proteins with either a Q174K substitution within the ABC signature motif or an E193Q substitution within the Walker B motif (39). The pstB coding region (plus 310 nt upstream and 650 nt downstream) was PCR amplified as two fragments; the middle primers overlapped by 20 nt, and the desired mutation was incorporated into the overlap region of the primers (oligonucleotides for Q174K, DF083/DF084 and DF085/DF086; oligonucleotides for E193Q, DF083/DF087 and DF088/DF089).…”

Structure, Function, and Evolution of Bacterial ATP-Binding Cassette Systems

“…Thiol-specific reagents react more readily with a Cys in one ABC in the histidine transporter than with a Cys in the other, suggesting that structural asymmetry may exist before the binding of nucleotide (257). In the maltose transporter, asymmetries are seen in the pattern of cross-linking between cysteines placed in the helical domains of the ABC MalK and the IM proteins MalF and MalG (92,216). In both cases, the IM region is heterodimeric, which could contribute to differences.…”

Dynamics of α-helical subdomain rotation in the intact maltose ATP-binding cassette transporter

“…We detected changes in SL mobility at position 168 in the RecA-like subdomain in response to nucleotide-binding even though it is 20 Å from the site of nucleotide-binding. Although the EAA loops move along with MalK during the catalytic cycle, differences in cross-linking between EAA loops and Q-loops are seen during the catalytic cycle of MalFGK 2 (44).…”