Characterizing ligand and lipid modulation of function in membrane transporters

The cellular membrane is a fundamental component of a living cell, facilitating essential processes such as selective material transport and information exchange with the environment, mediated by membrane-associated proteins. The diversity of lipid composition in biological membranes and its impact on the structure, dynamics, and function of membrane proteins are now well-established. However, experimentally characterizing these crucial lipid-protein interactions remains highly challenging. Molecular dynamics (MD) simulations provide a powerful complementary method, offering sufficient temporal and spatial resolution to obtain atomic-level structural and energetic details of lipid-protein interactions. Using MD simulations to explore lipid-protein interactions and characterize lipid-influenced protein structure and dynamics, can shed light on the mechanisms of membrane protein function. ABCG2 is a critical member of the ATP-binding cassette (ABC) transporter family, playing a vital role in extruding a wide range of xenobiotics and drugs from the cell. Overexpression of ABCG2 has been linked to multi-drug resistance in cancer cells, leading to the failure of chemotherapy treatments. Thus, understanding the structural and functional properties of ABCG2 is crucial for developing effective cancer therapies. Here, we study the binding dynamics of topotecan and tariquidar bound to ABCG2. Recently, the structural characterization of ABCG2 in complex with the anti-cancer drug topotecan has shed light on the conformational changes associated with drug binding and transport. Our collaborator’s in the Locher lab from ETH, have taken this investigation further by presenting cryo-EM structures of ABCG2 in the presence of a slow substrate and a special modulator called tariquidar. The new structures reveal that tariquidar induces two distinct conformations of ABCG2 under turnover conditions, which are similar to those observed with topotecan. To gain further insights into the molecular mechanism of drug binding and transport, we conducted molecular dynamics simulations of drug-bound and apo ABCG2 in native-like lipid bilayers. Our simulations revealed that the size of the ligand is a major determinant of its binding stability. The smaller drug, topotecan, exhibits a highly dynamic binding mode, where it interacts directly with the phospholipid molecules in the binding pocket. In contrast, the larger tariquidar occupies most of the available space in the pocket, making it difficult for the lipids to penetrate and interact with it. Interestingly, we also observed the spontaneous penetration of phospholipids into the binding cavity of ABCG2 in the apo inward-open state. This finding suggests a putative general path for hydrophobic and amphiphilic substrates to be recruited directly from the membrane to the binding pocket of ABCG2. Intriguingly, we also found that ABCG2 does not accommodate cholesterol as a substrate, which is omnipresent in plasma membranes that contain ABCG2. Moreover, we found cholesterol to act as a transient gate-keeper and prohibit the penetration of phospholipids into ABCG2’s lumen. Considering the necessity of cholesterol for the transport function of ABCG2 reported in numerous experimental studies, our simulations hint on one of the ways cholesterol can help the transport function in ABCG2; that is through keeping its mechanism free of interference from the phospholipids. Overall, this study provides an understanding of the molecular mechanisms underlying drug binding and transport by ABCG2. These findings have significant implications for the development of new cancer therapies and the design of drugs that can evade the efflux activity of ABCG2.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biophysics & Quant Biology

Grantor

University of Illinois Urbana-Champaign

Year dc:date

2025

Author dc:creator

• Rasouli, Ali

Contributors dc:contributor

• Tajkhorshid, Emad
• Shukla, Diwakar
• Aksimentiev, Aleksei
• Pogorelov, Taras

• Membrane transport
• Molecular dynamics

Statement dc:rights

• Copyright 2025 Ali Rasouli

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/129491

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F129491