Insights into dynamics of membrane transporters from computational techniques

Membrane transport proteins are the molecular machinery that selectively regulates transmembrane traffic of a broad spectrum of solutes. A major class of membrane transporters, secondary active transporters, uses the downhill electrochemical gradient of one solute as the energy source to power the uphill transport of another. The central questions underlying the secondary active transport mechanism are the coupling between energy sources and protein dynamics, and how it affects the binding and translocation of the driven substrate. Characterizing the molecular events at a microscopic level is the key to understanding the underlying transport mechanism. Computational tools, most prominently molecular dynamics (MD) simulations, are proven powerful to investigate a wide range of involved dynamical events. This dissertation uses and develops computational tools to examine multiple critical aspects of membrane transporters, with a focus on an important drug target, the serotonin transporter (SERT). SERT belongs to the family of neurotransmitter:sodium symporters (NSSs). It terminates serotonin signaling by recycling excess serotonin from the synaptic gaps to presynaptic neurons, and the malfunction of SERT is related to many psychological diseases. The first part of this dissertation focuses on deciphering the ion/substrate-coupling mechanism of SERT. SERT imports serotonin with Na+ through a conformational transition from an outward-facing (OF) state to an inward-facing (IF) state. Binding of Na+ locks SERT in an OF state, whereas subsequent binding of serotonin unlocks it and promotes the OF-to-IF transition, the molecular mechanism of which remains unknown. Using MD simulations and free energy calculations, the bound serotonin is identified to induce unique gating dynamics compared to other ligand-loading states, which further lowers the energy barrier along the OF-to-IF transition pathway to promote the serotonin import. The second study on SERT characterizes the cytosolic K+ binding to IF SERT, a kinetic decision point that frustrates the outward transport of serotonin and triggers the returning transition from IF to OF state. With an extensive set of MD simulations, K+ is found to bind to a cation-binding site and contract the cytosolic entry to the protein, which is not observed when Na+ is at the same site, shedding light on the ion-dependence of the IF-to-OF transition. Lastly, in collaboration with a cryo-electron microscopy (cryo-EM) study, we investigated the binding of ibogaine, a non-competitive inhibitor, to SERT in multiple functional states. We developed a computational docking approach to systematically search for the optimal ibogaine-binding poses, which simultaneously fit the experimental data and establish favorable interactions with the protein. The optimal binding poses in all three states share a salt bridge between an aspartate residue and the charged ammonium group of the ligand, a hallmark of substrate binding conserved among NSSs. Together with the cryo-EM work, the consistent ibogaine-binding modes suggest that ibogaine binding may enable isomerization of SERT, and the conformational preference of the ibogaine-bound SERT may explain the non-competitive inhibition of ibogaine. The works on membrane transporters triggered the development of two new computational tools. The first tool is Genetic Optimization of Ligand in Experimental Maps (GOLEM). This automated and robust ligand docking method models the ligand's optimal pose and conformation in cryo-EM maps efficiently and reliably. GOLEM employs a Lamarckian genetic algorithm to efficiently explore the ligand's conformational, orientational, and positional space, with explicit consideration of water displacement and bridging water molecules' position and orientation. With these features, GOLEM is a valuable tool for ligand modeling in cryo-EM efforts toward drug discoveries. The other tool identifies long-range hydrogen-bonding connections through water wires. It can be used in determining the proton transition pathway, channel connectivity, etc. Both tools are implemented as an extension in the widely used program Visual Molecular Dynamics (VMD) for easier access and visualization.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biophysics & Quant Biology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2022

Author dc:creator

• Zhao, Zhiyu

Contributors dc:contributor

• Tajkhorshid, Emad
• Grosman, Claudio
• Shukla, Diwakar
• Zhang, Kai

• membrane transporter
• serotonin transporter
• molecular dynamics simulation
• ligand docking

Statement dc:rights

• Copyright 2022 Zhiyu Zhao

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/115734

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F115734