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Biochemistry. 2010 Feb 16;49(6):1105-14. doi: 10.1021/bi901412a.

Simulation of spontaneous substrate binding revealing the binding pathway and mechanism and initial conformational response of GlpT.

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Department of Biochemistry, College of Medicine, Center for Biophysics and Computational Biology, and Beckman Institute for Advanced Science and Technology, University of Illinois, Urbana, Illinois 61802, USA.


Glycerol 3-phosphate transporter (GlpT) mediates the import of glycerol 3-phosphate (G3P) using the gradient of inorganic phosphate (P(i)). To study the process and mechanism of substrate binding and to investigate the protein's initial response, we performed equilibrium simulations of wild-type GlpT and several of its mutant forms in membranes in the presence of all physiologically relevant substrates (P(i)(-), P(i)(2-), G3P(-), and G3P(2-)). The simulations capture spontaneous substrate binding of GlpT, driven by the positive electrostatic potential of the lumen. K80 is found to act as a "hook" making the first encounter with the substrate and guiding it toward the binding site, where it binds tightly to R45, a key binding site residue that acts as a "fork" holding the substrate. R269 establishes no direct contact with the substrate during the simulations, a surprising behavior given its structural pseudosymmetry to R45. In all substrate-bound systems, partial closing of the cytoplasmic half of GlpT was observed. The substrate appears to stabilize the partially occluded state, as in the two apo simulations either no closing was observed or the protein reverted to its open form toward the end of the simulation, whereas in all substrate-bound systems, a stable partially closed state was produced. Along with the modulation of the periplasmic salt bridge network, these substrate-induced events destabilize the periplasmic half while inducing a closure in the cytoplasmic half, thus capturing the early stages of the proposed rocker-switch mechanism in GlpT.

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