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The Substrate Import Mechanism of the Human Serotonin Transporter

preprint
revised on 09.04.2020 and posted on 10.04.2020 by Matthew Chan, Balaji Selvam, Heather Young, Erik Procko, Diwakar Shukla

The serotonin transporter, SERT, initiates the reuptake of extracellular serotonin in the synapse to terminate neurotransmission. Recently, the cryo-EM structures of SERT bound to ibogaine resolved in different states provided the first glimpse of functional conformations at atomistic resolution. However, the conformational dynamics and structural transitions to various intermediate states are not fully understood. Furthermore, while experimental SERT structures were complexed with drug molecules and inhibitors, the molecular basis of how the physiological substrate, serotonin, is recognized, bound, and transported remains unclear. In this study, we performed microsecond long simulations of the human SERT to investigate the structural dynamics to various intermediate states and elucidated the complete substrate import pathway. Using Markov state models, we characterized a sequential order of conformational driven ion-coupled substrate binding and transport events and calculated the free energy barriers of conformation transitions associated with the import mechanism. We identified a set of crucial residues that recognize the substrate at the extracellular surface of SERT and our biochemical screening results show that mutations causes dramatic reduction in transport function. Our simulations also revealed a third sodium ion binding site coordinated by Glu136 and Glu508 in a buried cavity which helps maintain the conserved fold adjacent to the orthosteric site for transport function. The mutation of these residues results in a complete loss of transport activity. Our study provides novel insights on the molecular basis of dynamics driven ion-substrate recognition and transport of SERT that can serve as a model for other closely related neurotransmitter transporters.

Funding

NSF MCB 18-45606, NIH R21 MH113155

History

Email Address of Submitting Author

diwakar@illinois.edu

Institution

University of Illinois at Urbana-Champaign

Country

United States of America

ORCID For Submitting Author

0000-0003-4079-5381

Declaration of Conflict of Interest

No conflict of Interest

Version Notes

Additional simulations and analysis performed in this revised version.

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