Application of the String and 2D Hamiltonian Replica Exchange Umbrella Sampling Methods for the Study of Conformational Changes in the Bacterial Aspartate Transporter Glt(Ph)

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Glt(Ph) is a homotrimeric protein involved in the sodium coupled transport of aspartate through the cellular membrane of Pyrococcus horikoshii. It belongs to the solute carrier 1 (SLC1) family, which comprises of a variety of proteins responsible for the trans-membrane transport of acidic or neutral amino acids in prokaryotes and eukaryotes. An important member of this family is the human glutamate transporter, which regulates the concentration of the neurotransmitter glutamate in the central nervous system. Unfortunately, its crystal structure is currently unknown. As Glt(Ph) has well resolved crystal structures in different functionally significant conformations, it has been studied extensively as a structural and functional analogue of the human glutamate transporter. Previous experimental and molecular dynamics studies have shown that substrate binding and the presence or absence of a lipid bilayer induce considerable conformational changes in Glt(Ph) which have been linked to the aspartate movement through the membrane. In this work two different sampling methods - 2D Hamiltonian Replica Exchange Umbrella Sampling (2DHREUS) and the String method - are used for the evaluation of potentials of mean force of a gate movement in Glt(Ph) associated with substrate binding to the active site. The gate opening has a low energy barrier and can be observed in the apo state even in the absence of a substrate. The String method compares well to the results of our benchmark 2DHREUS calculations and captures the most important structural and thermodynamical aspects of the conformational changes.