An Insight into the XylE Transport Cycle by Characterizing Proton Binding Site and its Coupling to the Substrate Transport

Abstract

Characterizing the proton binding site and its coupling to the substrate transport??in XylE?The diffusion of glucose across the plasma membrane is facilitated by carrier proteins called GLUTs. In the absence of a crystal structure for GLUTs, XylE serves as an excellent model to study the transport cycle in these proteins, not only due to its high sequence and structural similarity, but since it is the only MFS member with structures in various conformational states. In this study, we aim to characterize the structural determinants of substrate and proton binding and their coupling to protein conformational transitions in XylE. Using multiple sets of equilibrium MD simulations and free energy perturbation (FEP) calculations, we propose one of the acidic residues of XylE within the transmembrane region of the protein as the uncharacterized proton-binding site. Through comparative simulation studies, we show that the protonation of this residue results in the breakage of a salt bridge, which in turn induces the transition of the protein from the occluded state towards the open state. The observed conformational coupling, which was reproduced in multiple independent simulations, appears to involve the rotation of TM2 and TM7, and thereby perturbing the Xylose binding site as evident by the movement of Y298. Using sequence analysis of GLUTs we show the absence of this mechanistically important salt bridge in GLUTs, which can account for their H+-independent transport mechanism. This work hence claims to give insight into the thermodynamic cycle corresponding to the change from outward facing occluded state to the open state and hence helps in elucidating the entire cycle in future.