# Visualizing the Functional Dynamics of P-Glycoprotein and Its Modulation by Elacridar via High-Speed Atomic Force Microscopy

## TL;DR

This study uses high-speed atomic force microscopy to visualize how P-glycoprotein moves and how Elacridar affects its motion, offering new insights into drug resistance in cancer.

## Contribution

The study is the first to directly visualize the dynamic conformational changes of P-glycoprotein and how Elacridar modulates these motions.

## Key findings

- P-glycoprotein exhibits large-scale spontaneous motions in its apo state.
- Elacridar at low concentrations activates P-glycoprotein dynamics, while high concentrations rigidify it.
- High-speed atomic force microscopy reveals structural dynamics linked to drug resistance mechanisms.

## Abstract

P-glycoprotein (P-gp) is an ATP-driven transporter that effluxes a wide range of xenobiotics from cells, and its overexpression is a primary cause of multidrug resistance (MDR) in cancer. It is well-established that P-gp functions through conformational changes, yet its large-scale structural dynamics at work have been unexplored. Here, we directly visualized single P-gp molecules reconstituted in nanodiscs using high-speed atomic force microscopy (HS-AFM). The HS-AFM movies revealed that P-gp is intrinsically dynamic in its apo state, with its nucleotide-binding domains (NBDs) undergoing large, spontaneous opening and closing motions. However, addition of ATP stabilized a conformation characterized by NBD proximity with a strong tendency toward closure. We then leveraged this dynamic viewpoint to elucidate the relationship between Elacridar’s function and the resulting structural dynamics of P-gp. Elacridar is designed to overcome multidrug resistance (MDR) in cancer and acts as a potent dual inhibitor of both P-gp and the Breast Cancer Resistance Protein (BCRP), effectively blocking the drug efflux function of these transporters. This inhibitor has suggested concentration-dependent function: it is effluxed as a substrate at low concentrations and acts as an inhibitor at high concentrations. Our direct observations revealed that low concentrations induced active dynamics in P-gp, whereas high concentrations severely restricted its motion, leading to a rigid, non-productive state. Our study provides critical insights into how observing molecular motion itself can unravel complex biological mechanisms.