The Detergent-Soluble Maltose Transporter Is Activated by Maltose Binding Protein and Verapamil

Abstract

The maltose transporter FGK2 complex of Escherichia coli was purified with the aid of a glutathione S-transferase molecular tag. In contrast to the membrane-associated form of the complex, which requires liganded maltose binding protein (MBP) for ATPase activity, the purified detergent-soluble complex exhibited a very high level of ATPase activity. This uncoupled activity was not due to dissociation of the MalK ATPase subunit from the integral membrane protein MalF and MalG subunits. The detergent-soluble ATPase activity of the complex could be further stimulated by wild-type MBP but not by a signaling-defective mutant MBP. Wild-type MBP increased the V max of the ATPase 2.7-fold but had no effect on the K m of the enzyme for ATP. When the detergent-soluble complex was reconstituted in proteoliposomes, it returned to being dependent on MBP for activation of ATPase, consistent with the idea that the structural changes induced in the complex by detergent that result in activation of the ATPase are reversible. The uncoupled ATPase activity resembled the membrane-bound activity of the complex also with respect to sensitivity to NaN 3 , as well as a mercurial, p-chloromercuribenzosulfonic acid. Verapamil, a compound that activates the ATPase activity of the multiple drug resistance P-glycoprotein, activated the maltose transporter ATPase as well. The activation of this bacterial transporter by verapamil suggests that a structural feature that is conserved among both eukaryotic and prokaryotic ATP binding cassette transporters is responsible for this activation.The membrane components of the maltose transport system of Escherichia coli catalyze the intracellular accumulation of malto-oligosaccharides at the expense of ATP hydrolysis (5). They comprise two integral membrane proteins, MalF and MalG, and two copies of a cytoplasmic ATP binding subunit, MalK (36). In addition to these components directly involved in substrate translocation, there is a periplasmic maltose binding protein (MBP) and a maltoporin (LamB) located in the outer membrane (5). This transporter is a member of the ATP binding cassette (ABC) superfamily, and the MalK sequence shares significant similarity with the sequences of several other prokaryotic and eukaryotic transport proteins that belong to this family.The FGK2 complex has been extensively characterized both genetically and biochemically. In most instances, the transporter has been studied either in whole cells, in subcellular vesicles, or in some cases following purification and reconstitution, in proteoliposomes. In all cases, the transport and ATPase activities of the FGK2 complex have been shown to be strongly dependent on MBP. Efforts to study the molecular basis of these transport and ATPase activities have been limited by the need to reconstitute the transporter in proteoliposomes. It has been generally assumed that a detergent-soluble form of this and other ABC transporters would not be a useful model for studying their molecular properties. Indeed, several publications have reported...

ATP Alone Triggers the Outward Facing Conformation of the Maltose ATP-binding Cassette Transporter

“…Instead, we find that ATP alone is sufficient. Because MalE does not facilitate the binding of ATP to the transporter (16,17), nor the outward facing conformation, the results imply that MalE must stimulate a step occurring during the return of the transporter to the inward facing state.…”

Both maltose-binding protein and ATP are required for nucleotide-binding domain closure in the intact maltose ABC transporter

“…Both AMP-PNP-Mg 2ϩ and maltose-MBP were required to close the interface. Because the ATPase activity of the transporter in the absence of MBP is much lower in proteoliposomes (Table 1) than in detergent (24), it was also possible to demonstrate the failure of ATP-Mg 2ϩ to stabilize the closed conformation of the reconstituted trans- porter in the absence of MBP (Fig. 5B).…”

Insights into ABC Transport in Archaea

“…Finally, the entire transporter was heterologously expressed in E. coli and purified . The solubilized complex showed intrinsic ATPase activity, but the ATPase was not further stimulated by the addition of TMBP as observed for the bacterial system (Reich-Slotky et al, 2000). The lack of stimulation might be explained by the difficulties to find detergents which are not precipitating at high temperatures and the absence of archaeal ether-lipids which have been shown to increase the stability of archaeal membrane proteins (Elferink et al, 1992).…”