Regulation mechanism and substrate specificity of the Escherichia coli maltose transporter

Shanshuang Chen, Purdue University

Abstract

Efficient carbon uptake and metabolism are essential for life. When multiple carbon sources are available, bacteria selectively utilize specific carbon sources in order to optimize carbon uptake from a dynamic environment. Discrimination among carbon sources is achieved by a mechanism known as inducer exclusion, where utilization of the preferred carbon source inhibits the uptake and/or metabolism of others. The glucose-specific enzyme IIA (EIIAGlc) from the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) has been found to play a key role in inducer exclusion. The uptake of the metabolically preferred carbon source, glucose, by PTS elevates the level of unphosphorylated EIIAGlc, which inhibits uptake of several non-PTS carbon sources by binding directly to their transporters. In this dissertation, we report the crystal structure of unphosphorylated EIIAGlc in complex with the Escherichia coli maltose transporter, a member of the ATP-binding cassette (ABC) transporter superfamily. The crystal structure shows that EIIAGlc allosterically inhibits maltose import by stabilizing the maltose transporter in a conformation similar to the previously determined inward-facing, resting state conformation. Also, we identified that the N-terminal 18 residues possibly function as a membrane-anchor to increase the effective EIIAGlc concentration at the membrane. Based on biochemical and structural studies, the large, extended periplasmic P2 loop of MalF, which is unique to the maltose transporter, was postulated to be important in the recognition of the periplasmic maltose binding protein (MBP) and its recruitment to the membrane surface. MBP is responsible not only for the acquisition and delivery of maltodextrins to the transporter, but also for the stimulation of the transporter ATPase activity. Thus, the interaction between MBP and the transporter in the periplasm must transmit a signal across the membrane for the activation of the cytoplasmic ATPase. Crystal structures of the MBP-transporter complex revealed two distinct MBP-transporter interfaces, one of which is formed by MBP and the P2 loop. In this dissertation, we characterize the role of the P2-MBP interaction in signal transmission, using a transporter construct where MBP is covalently linked to the P2 loop. We show that the interaction between MBP and the P2 loop is maintained throughout the transport cycle. Furthermore, we demonstrate that the presence of maltose is not necessary for the MBP-P2 interaction, but is required for the stimulation of the transporter ATPase. Therefore, the interaction between MBP and the P2 loop is insufficient for signaling substrate availability across the membrane. In cooperation with a coworker, Dr. Michael Oldham, we also uncovered the structural basis for the substrate specificity of the maltose transport system. The maltose transport system can only transport linear maltodextrins ranging in size from maltose to maltoheptaose. By determining the crystal structures of the maltose transporter in two distinct conformational states in the presence of large maltodextrins, we observed that in the pre-translocation state, MBP and MalG recognize four glucosyl units from the reducing end, whereas in the outward-facing state, the transmembrane binding site formed by MalF recognizes three glucosyl units from the non-reducing end. These structures explain how specificity to physiological substrates is achieved, and why modified or oversized maltodextrins are not substrates for the maltose transport system.

Degree

Ph.D.

Advisors

Cramer, Purdue University.

Subject Area

Molecular biology|Biochemistry|Biophysics

COinS