Binding of the immunomodulatory drug Bz-423 to mitochondrial FoF1-ATP synthase in living cells by FRET acceptor photobleaching

TL;DR

This study demonstrates the binding of the immunomodulatory drug Bz-423 to mitochondrial FoF1-ATP synthase in living yeast cells using FRET acceptor photobleaching, revealing insights into its interaction at the molecular level.

Contribution

It introduces a novel application of FRET acceptor photobleaching microscopy to visualize drug binding to mitochondrial enzymes in living cells.

Findings

Bz-423 binds specifically to the top of the F1 domain of FoF1-ATP synthase.

Superresolution imaging revealed mitochondrial structure in living yeast.

FRET confirmed drug binding in vivo.

Abstract

Bz-423 is a promising new drug for treatment of autoimmune diseases. This small molecule binds to subunit OSCP of the mitochondrial enzyme FoF1-ATP synthase and modulates its catalytic activities. We investigate the binding of Bz-423 to mitochondria in living cells and how subunit rotation in FoF1-ATP synthase, i.e. the mechanochemical mechanism of this enzyme, is affected by Bz-423. Therefore, the enzyme was marked selectively by genetic fusion with the fluorescent protein EGFP to the C terminus of subunit gamma. Imaging the threedimensional arrangement of mitochondria in living yeast cells was possible at superresolution using structured illumination microscopy, SIM. We measured uptake and binding of a Cy5-labeled Bz-423 derivative to mitochondrial FoF1-ATP synthase in living yeast cells using FRET acceptor photobleaching microscopy. Our data confirmed the binding of Cy5-labeled Bz-423 to the top of the F1 domain of the enzyme in mitochondria of living Saccharomyces cerevisiae cells.