Path Searching Towards the Symmetric Inward Open Structure of LeuT

Abstract

Few crystal structures are available for transporters related to the biomedically important neurotransmitter transporters, such as the serotonin and dopamine transporters. One of these structures is the bacterial analog LeuT, which transports Leucine. Until recently the available crystal structures of LeuT provided information on only two distinct states: an outward open state, where the S1 substrate-binding site is exposed to the extracellular vestibule, and an occluded state, where the S1 binding site is occluded from both the extracellular and intracellular vestibules. Therefore, there was not enough structural information to support an understanding of the substrate transport mechanism. Several computational models emerged to describe the inward facing conformation of LeuT in which the substrate is released. Given the structural information, some of these models (e.g., the symmetric inward open model - SIO) assumed rigid body motions in which the molecule would transition between the outward open-occluded-inward open states. We attempt to evaluate the feasibility of various models of LeuT by generating a transition path between distinct conformational states using the Motion Planning (MP) module Pathrover, a method that can identify a set of low-energy, clash-free structural intermediates between known end states. Because the sodium-hydantoin transporter Mhp1, which transitions between the same kind of states, has been crystallized in three distinct conformations, and intermediates have been calculated from a force-based approach, dynamic importance sampling (DIMS), it was used here to test the robustness of the Pathrover approach. For Mhp1 we find a clear overlap between the Pathrover computed intermediates and the DIMS intermediates, and conclude that the transition among the Mhp1 states is well represented by Pathrover. The intermediates calculated for LeuT in this work form the basis for comprehensive molecular dynamics simulations probing the molecular mechanism.