Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Dr. Brian Shilton

Abstract

This thesis investigates the mechanism of activity-coupling in the maltose transporter of Escherichia coli (MalFGK2); the way ATP hydrolysis is prevented in the absence of maltose, and then enabled to drive maltose transport. Like other ATP binding cassette importers, MalFGK2 requires substrate to be presented by a peripheral substrate-binding protein, in this case the maltose binding protein (MBP). MBP predominantly adopts an ‘open’ resting state, but undergoes a rotation of its two domains to a ‘closed’ state after maltose binding. In the closed state MBP is able to activate MalFGK2 to stimulate ATP hydrolysis and maltose transport.

Engineered mutants of MBP have been used to test the responsiveness of the transporter to changes in the conformational state of its binding protein. Careful analysis of ATPase stimulus by wild type MBP indicates that the open state of MBP is able to bind the transporter and promote ATP hydrolysis in the absence of maltose. Another mutant MBP, able to adopt the closed state in the presence of either sucrose or maltose, demonstrates that ATP hydrolysis is activated by interactions between the transporter and binding protein, and not between the transporter and substrate. Further, using MBP mutants incorporating introduced disulfide bonds and inter-domain cross-linkers we show that the closed form of MBP is able to activate substantial, but not maximal, ATPase activity without adopting the open conformation or releasing its bound substrate.

It has been determined that both stable forms of MBP, maltose-bound-closed and unliganded-open, separately stimulate ATP hydrolysis from the transporter. In contrast, the substrate itself does not directly stimulate activity but instead activates hydrolysis by stabilizing the closed state of MBP. Taken together our data indicate that the transporter exists in equilibrium between an inactive resting state and conformations receptive to binding by the closed and open forms of MBP. This suggests that uncoupled ATP hydrolysis is prevented by destabilizing ATPase relevant conformations of the transporter, and that MBP activates hydrolysis by binding these conformations in its closed and open states.

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