Structure and Dynamics Study of LeuT Using the Markov State Model and Perturbation Response Scanning Reveals Distinct Ion Induced Conformational States

The
bacterial leucine transporter (LeuT), a close homologue of
the eukaryote monoamine transporters (MATs), currently serves as a
powerful template for computer simulations of MATs. Transport of the
amino acid leucine through the membrane is made possible by the sodium
electrochemical potential. Recent reports indicate that the substrate
transport mechanism is based on structural changes such as hinge movements
of key transmembrane domains. In order to further investigate the
role of sodium ions in the uptake of leucine, here we present a Markov
state model analysis of atomistic simulations of lipid embedded LeuT
in different environments, generated by varying the presence of binding
pocket sodium ions and substrate. Six metastable conformations are
found, and structural differences between them along with transition
probabilities are determined. We complete the analysis with the implementation
of perturbation response scanning on our system, determining the most
sensitive and influential regions of LeuT, in each environment. Our
results show that the occupation of sites Na1 and Na2, along with
the presence of the substrate, selectively influences the geometry
of LeuT. In particular, the occupation of each site Na1/Na2 has strong
effects (in terms of changes in influence and/or sensitivity, as compared
to the case without ions) in specific regions of LeuT, and the effects
are different for simultaneous occupation. Our results strengthen
the rationale and provide a conformational mechanism for a putative
transport mechanism in which Na2 is necessary, but may not be sufficient,
to initiate and stabilize extracellular substrate access to the binding
pocket.