Unliganded maltose-binding protein triggers lactose transport in an Escherichia coli mutant with an alteration in the maltose transport system

Abstract

Escherichia coli accumulates malto-oligosaccharides by the maltose transport system, which is a member of the ATP-binding-cassette (ABC) superfamily of transport systems. The proteins of this system are LamB in the outer membrane, maltose-binding protein (MBP) in the periplasm, and the proteins of the inner membrane complex (MalFGK 2 ), composed of one MalF, one MalG, and two MalK subunits. Substrate specificity is determined primarily by the periplasmic component, MBP. However, several studies of the maltose transport system as well as other members of the ABC transporter superfamily have suggested that the integral inner membrane components MalF and MalG may play an important role in determining the specificity of the system. We show here that residue L334 in the fifth transmembrane helix of MalF plays an important role in determining the substrate specificity of the system. A leucine-to-tryptophan alteration at this position (L334W) results in the ability to transport lactose in a saturable manner. This mutant requires functional MalK-ATPase activity and the presence of MBP, even though MBP is incapable of binding lactose. The requirement for MBP confirms that unliganded MBP interacts with the inner membrane MalFGK 2 complex and that MBP plays a crucial role in triggering the transport process.The maltose transport system of Escherichia coli mediates the unidirectional transport of ␣(1-4)-linked malto-oligosaccharides (up to maltoheptaose) from the extracellular medium into the cytoplasm of the cell (14). It is a member of the ATP-binding-cassette (ABC) superfamily of transport systems (2) by virtue of its strong sequence homology and similar domain structure. Members of this family are found in a variety of organisms and include the cystic fibrosis transmembrane conductance regulator in humans as well as the STE6 protein of Saccharomyces cerevisiae (24). The maltose transport system is composed of five proteins: LamB (maltoporin), maltosebinding protein (MBP), MalF, MalG, and MalK (44). The genes that encode these proteins are located in divergent operons in the malB region of the E. coli chromosome.The LamB protein is an outer membrane porin that facilitates the diffusion of malto-oligosaccharides into the periplasm of the cell (39, 47). LamB also functions as the receptor for phage (36). MBP is a soluble periplasmic protein that binds malto-oligosaccharides with a high affinity (K d ϭ 0.1 to 1 M) (15,28,40). MBP is thought to deliver the substrate to the inner membrane complex, and it is required for the transport of maltose (41, 50). The three-dimensional structure of MBP has been determined to the atomic level by X-ray crystallography (46). The subunit stoichiometry of the inner membrane MalF-MalG-MalK complex is 1:1:2 (MalFGK 2 ) (9). This complex transports the substrate across the inner membrane in an ATP-dependent manner. The MalF and MalG proteins are integral membrane proteins that are predicted to traverse the membrane eight and six times, respectively (3,8,16). MalK is a peripheral protein associ...

A Member of the Second Carbohydrate Uptake Subfamily of ATP-Binding Cassette Transporters Is Responsible for Ribonucleoside Uptake inStreptococcus mutans

“…Deka et al (5) reported that PnrA, a lipoprotein in Treponema pallidum, binds purine ribonucleosides (adenosine, guanosine, inosine, 2-deoxyadenosine, and 2-deoxyguanosine) and, to a lesser extent, pyrimidine ribonucleosides (cytidine and uridine). Although such binding of substrates by a solute binding protein is strongly indicative that they are transported by the associated ABC transporter, this is not necessarily so, as it is known that the membrane components of ABC transporters contribute to their specificity (18,32). Unfortunately, the genetic intransigence of T. pallidum hinders further characterization of pnrA or the adjacent ABC transporter genes.…”

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

“…MBP was purified by affinity chromatography as described before (24). Briefly, fresh transformants of HS3309 were grown for 18 h in Terrific Broth medium (12 g of tryptone per liter, 24 g of yeast extract per liter, 0.4% glycerol, 2.31 g of KH 2 PO 4 , 12.54 g of K 2 HPO 4 per liter), and the periplasmic fraction was loaded onto an amylose column.…”

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

“…The sucrose-binding MBP (sMBP) enables us to present the maltose transporter with either maltose or sucrose in equivalent contexts and distinguish whether the substitution influences the ATPase activity of MalFGK 2 . Sucrose is a good alternative substrate for this purpose because experiments by Shuman and co-workers (13,14) have shown that it has a very poor ability to compete for the maltose-binding site in MalFGK 2 , indicating that the change in sugar structure is sufficient to disrupt specific binding interactions with MalFGK 2 .…”