Conformational Transition Pathway of GkPOT

Abstract

GkPOT, a Proton-Coupled coupled Oligopeptide Transporter transporter (POTs) from Geobacillus Kaustophilus, is among the symporter members of the Major Facilitator Superfamily (MFS) of transporters. Functional and structural analysis of various POTs suggest that the uptake of small peptides and peptide- like molecules to the cell across the cell membrane, via the alternating access mechanism, utilizes an inward directed proton electrochemical gradient the source of energy for their active symport function. The transition between the inward-facing (IF) and outward-facing (OF) conformations is important in understanding the structural relationship between large-scale protein conformational changes and substrate translocation that has often times remained elusive due to the short time scales of molecular dynamics simulations (MD) limitations of computational and experimental effort techniques. Using an extensive set of enhanced sampling techniques, we have characterized the conformational transition pathway of GkPOT. We have aimed at describing the large-scale conformational changes between the IF and OF states while in the presence and absence of substrate (dialanine peptide). Nonequlibrium pulling simulations were initiated from the known inward facing IF conformation and steered the system toward the unknown outward facing OF conformation using orientation based collective variables defined on different transmembrane helices. The transition pathways are far from equilibrium and are smoothed with string method with swarms of trajectories (SMwST), thus generating microseconds of biased and unbiased simlulations. Free energy profiles of the conformational transition pathways were obtained from a novel maximum-likelihood estimation method. Our results indicate that the presence of ligand significantly changes the conformational landscape with respect to the apo system which contains a greater degree of conformational variability.