Structural basis for substrate specificity in the Escherichia coli maltose transport system

Abstract

ATP-binding cassette (ABC) transporters are molecular pumps that harness the chemical energy of ATP hydrolysis to translocate solutes across the membrane. The substrates transported by different ABC transporters are diverse, ranging from small ions to large proteins. Although crystal structures of several ABC transporters are available, a structural basis for substrate recognition is still lacking. For the Escherichia coli maltose transport system, the selectivity of sugar binding to maltose-binding protein (MBP), the periplasmic binding protein, does not fully account for the selectivity of sugar transport. To obtain a molecular understanding of this observation, we determined the crystal structures of the transporter complex MBP-MalFGK2 bound with large malto-oligosaccharide in two different conformational states. In the pretranslocation structure, we found that the transmembrane subunit MalG forms two hydrogen bonds with malto-oligosaccharide at the reducing end. In the outward-facing conformation, the transmembrane subunit MalF binds three glucosyl units from the nonreducing end of the sugar. These structural features explain why modified malto-oligosaccharides are not transported by MalFGK2 despite their high binding affinity to MBP. They also show that in the transport cycle, substrate is channeled from MBP into the transmembrane pathway with a polarity such that both MBP and MalFGK2 contribute to the overall substrate selectivity of the system.

Keywords: maltodextrin; membrane protein.

Fig. 1.

The three components of the maltose transport system. Malto-oligosaccharides diffuse across the outer membrane through maltoporin, bind with MBP in the periplasm, and are transported across the inner membrane by the ABC transporter MalFGK₂.

Fig. 2.

Substrate specificity of the maltose transport system. (A) The maltose transport system takes up linear malto-oligosaccharides containing two to seven glucosyl units linked through the α-1, 4 bonds. The glucosyl unit with a free anomeric carbon (C1) is able to form an open chain with an aldehyde group through isomerism, and thus is defined as the reducing end (shown in red). The opposite end, the nonreducing end, is shown in green. To be consistent with the nomenclature used in the MBP studies, the glucosyl units are identified from the reducing end as g1, g2, g3, and so on. The structure of two large substrates, maltohexaose and maltoheptaose, are shown. (B) Examples of closely related carbohydrates that are not recognized by the maltose transport system.

Fig. 3.

Structure of the pretranslocation state in complex with maltoheptaose. (A) Ribbon representation and a surface slab view. The four glucosyl units of maltoheptaose are shown in stick models (red and gray). Color code: MBP, purple; MalG, yellow; MalF, blue; MalK, red and green. (B) Stereoview of the binding site. MBP is shown in a surface representation, the scoop loop of MalG is shown in a ribbon representation, and the glucosyl units are shown in stick models (red and gray) and as van der Waals dot surfaces (green). (C) Stereoview of the F_o-F_c map contoured at 2σ with the sugar molecule omitted in the phase calculation. (D) Stereoview of the detailed interactions between the sugar and residues in MBP and MalG. Hydrogen bonds are indicated by dashed lines.

Fig. 4.

Structure of the outward-facing conformation in complex with maltohexaose. (A) Ribbon representation and a surface slab view. Color code is as in Fig. 3. (B) Stereoview of the F_o-F_c map contoured at 3σ with the sugar molecule omitted in the phase calculation. (C) Stereoview of the binding site. MalF and MalG are shown in surface representations, and the glucosyl units are shown in stick models (red and gray) and as van der Waals dot surfaces (green). (D) Stereoview of the detailed interactions between the sugar and residues in MalF and MalG. Hydrogen bonds are indicated by dashed lines.

Fig. 5.

Substrate specificity is conveyed by both MBP and MalFGK₂. (A) Schematic model of substrate recognition in the pretranslocation state and the outward-facing state. The reducing end of the malto-oligosaccharide is in red, and the nonreducing end is in green. The glucosyl units bound by protein are shown as solid circles, and dashed circles represent the glucosyl units not observed in the crystal structures. (B) Stereoview of a modeled maltoheptaose bound to the pretranslocation state. The substrate-binding cavity at the interface of MBP and MalFGK₂ is shown as a mesh (cyan). (C) The ATPase of MalFGK₂ reconstituted into lipidic nanodiscs. M3, maltotriose; M6, maltohexaose, M7, maltoheptaose; M10, a mixture containing 65% maltodecaose, 24% maltononaose, 7% maltooctaose, 2% maltoheptaose, and 2% impurities. Data columns represent mean ± SD values from triplicate measurements.