Stimulation of the Maltose Transporter ATPase by Unliganded Maltose Binding Protein

Abstract

ATP hydrolysis by the maltose transporter (MalFGK 2 ) is regulated by maltose binding protein (MBP). Binding of maltose to MBP brings about a conformational change from "open" to "closed" that leads to a strong stimulation of the MalFGK 2 ATPase. In this study we address the long-standing but enigmatic observation that unliganded MBP is also able to stimulate MalFGK 2 . Although the mechanism of this stimulation is not understood, it is sometimes attributed to a small amount of closed (but unliganded) MBP that may exist in solution. To gain insight into how MBP regulates the MalFGK 2 ATPase, we have investigated whether the open or the closed conformation of MBP is responsible for MalFGK 2 stimulation in the absence of maltose. The effect of MBP concentration on the stimulation of MalFGK 2 was assessed: for unliganded MBP, the apparent K M for stimulation of MalFGK 2 was below 1 μM, while for maltose-bound MBP, the K M was approximately 15 μM. We show that engineered MBP molecules in which the open-closed equilibrium has been shifted towards the closed conformation have a decreased ability to stimulate MalFGK 2 . These results indicate that stimulation of the MalFGK 2 ATPase by unliganded MBP does not proceed through a closed conformation and instead must operate through a different mechanism than stimulation by liganded MBP. One possible explanation is that the open conformation is able to activate the MalFGK 2 ATPase directly.ATP Binding Cassette (ABC) transporters use the chemical energy of ATP hydrolysis to transport solutes across a membrane. ABC transporters can be divided into export systems and import systems, and a key question in both cases is the mechanism by which ATP binding and hydrolysis are regulated and coupled to substrate translocation. For ABC export systems, the substrate itself regulates the ATPase (1-3). ABC import systems, on the other hand, include a peripheral substrate binding protein, and the ATPase activity is regulated by interactions between the binding protein and the transmembrane components (4-6).The E. coli maltose transporter is a tractable and well-studied ABC import system consisting of the membrane associated complex MalFGK 2 and peripheral extracellular maltose binding protein (MBP). MalF and MalG are integral membrane proteins; MalK 2 is a dimer of ABC subunits bound to MalFG on the cytoplasmic side of the membrane. Structures of isolated MalK 2 have been solved and from these it is known that ATP binds in the dimer interface and promotes a tight association of the subunits, bringing each ATP molecule in contact with catalytic residues from the opposite subunit (7). Once ATP hydrolysis takes place, the subunits dissociate, allowing release of ADP and inorganic phosphate (8). In the intact MalFGK 2 †CORRESPONDING AUTHOR: bshilton@uwo.ca, Fax: 519-661-3175, Telephone: 519-661-4124. * current affiliation: Southern Crop Protection and Food Research Branch, Agriculture and Agrifood Canada, 1391 Sandford St., London, Ontario, Canada N5V 4T3 1 These authors contributed e...

Studies of the Maltose Transport System Reveal a Mechanism for Coupling ATP Hydrolysis to Substrate Translocation without Direct Recognition of Substrate

“…In addition, unliganded sMBP did not produce a significant increase in MalFGK 2 ATPase, in contrast to unliganded wtMBP that consistently produces a 2-fold stimulation (Fig. 1A) (11,15). Therefore, although the substitution of sucrose for maltose did not influence stimulation of the MalFGK 2 ATPase, when compared with wtMBP the mutations in sMBP have drastically compromised its overall ability to stimulate the MalFGK 2 ATPase.…”

“…Expression and Purification-Hexahistidine-tagged sMBP and wtMBP were expressed and purified from HS3309 (MalE Ϫ/Ϫ ) E. coli by Ni 2ϩ -affinity chromatography, removal of the affinity tag by tobacco etch virus protease cleavage, and ion exchange chromatography, as reported previously (15). Both proteins were denatured in 6 M guanidine and dialyzed exhaustively to remove trace sugars before being refolded by dropwise dilution and stored at Ϫ80°C in 50 mM Tris-HCl, pH 8 (15).…”

“…Both proteins were denatured in 6 M guanidine and dialyzed exhaustively to remove trace sugars before being refolded by dropwise dilution and stored at Ϫ80°C in 50 mM Tris-HCl, pH 8 (15).…”

“…Liposomes were prepared from Avanti TM crude E. coli phospholipids, and after homogenization by sonication, the liposomes were combined with MalFGK 2 -containing membranes by detergent dilution (15). The proteoliposomes were frozen at Ϫ80°C under N 2 until used.…”

“…Purified sMBP or wtMBP was added at various concentrations and in the presence or absence of 5 mM maltose or sucrose. ATP hydrolysis at 37°C was measured in vitro by assaying the appearance of inorganic phosphate, using ammonium molybdate, as described previously (15).…”