Abstract
The phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) is crucial for sugar uptake in bacteria. It has a membrane embedded component, EIIC, that translocates a sugar from the extracellular to the intracellular side of the cell. Before the sugar is released into the cytosol, a cytosolic EIIB protein binds to EIIC and transfers a phosphate group to the incoming sugar that prevents the sugar from escaping the cell and at the same time primes the sugar for entering metabolic cycles. Little is known concerning how EIICs recognize and transport carbohydrates or how an EIIC coordinates with EIIB to achieve phosphate transfer. Crystal structures of a maltose transporter bcMalT[1] and a N-diacetylchitobiose transporter bcChbC[2] were solved recently, and the two structures appear to be in different states of a transport cycle: bcMalT in an outward facing state and bcChbC in an inward facing state. The bcChbC structure provides a template to build a model of bcMalT in an inward facing state, and vise versa. To examine the models, we designed pairs of cysteine residues that are distant in the crystal structures but are predicted to move close to each other in the alternate conformation. Several pairs of cysteines in in both bcMalT and bcChbC can be crosslinked by micromolar concentrations of mercury, indicating that these residues can move close to each other. We then solved the structure of the T280C/E54C bcMalT double cysteine mutant in the crosslinked state to 3.6 A resolution, and the structure is indeed in an inward-facing conformation. The new structure illustrates the large-scale movement of a structurally conserved domain in bcMalT, and shows that EIIC employs an elevator-like mechanism for substrate translocation. Further analyses suggest how the inward facing conformation could interact with an EIIB protein to achieve phosphate transfer. The structures also provide a solid starting point for investigating the dynamics of the EIIC protein using spectroscopic approaches.Reference[1]. Mccoy JG, Ren Z, Stanevich V, et al. The Structure of a Sugar Transporter of the Glucose EIIC Superfamily Provides Insight into the Elevator Mechanism of Membrane Transport. Structure. 2016;24(6):956-64.[2]. Cao Y, Jin X, Levin EJ, et al. Crystal structure of a phosphorylation-coupled saccharide transporter. Nature. 2011;473(7345):50-4.
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