Potent ClpP agonists with anticancer properties bind with improved structural complementarity and alter the mitochondrial N-terminome.

The mitochondrial ClpP protease is responsible for mitochondrial protein quality control through specific degradation of proteins involved in several metabolic processes. ClpP overexpression is also required in many cancer cells to eliminate reactive oxygen species (ROS)-damaged proteins and to sustain oncogenesis. Targeting ClpP to dysregulate its function using small-molecule agonists is a recent strategy in cancer therapy. Here, we synthesized imipridone-derived compounds and related chemicals, which we characterized using biochemical, biophysical, and cellular studies. Using X-ray crystallography, we found that these compounds have enhanced binding affinities due to their greater shape and charge complementarity with the surface hydrophobic pockets of ClpP. N-terminome profiling of cancer cells upon treatment with one of these compounds revealed the global proteomic changes that arise and identified the structural motifs preferred for protein cleavage by compound-activated ClpP. Together, our studies provide the structural and molecular basis by which dysregulated ClpP affects cancer cell viability and proliferation.

Functional Oligomeric Forms of Uncoupling Protein 2: Strong Evidence for Asymmetry in Protein and Lipid Bilayer Systems.

Stoichiometry of uncoupling proteins (UCPs) and their coexistence as functional monomeric and associated forms in lipid membranes remain intriguing open questions. In this study, tertiary and quaternary structures of UCP2 were analyzed experimentally and through molecular dynamics (MD) simulations. UCP2 was overexpressed in the inner membrane of Escherichia coli, then purified and reconstituted in lipid vesicles. Structure and proton transport function of UCP2 were characterized by circular dichroism (CD) spectroscopy and fluorescence methods. Findings suggest a tetrameric functional form for UCP2. MD simulations conclude that tetrameric UCP2 is a dimer of dimers, is more stable than its monomeric and dimeric forms, is asymmetrical and induces asymmetry in the membrane's lipid structure, and a biphasic on-off switch between the dimeric units is its possible mode of transport. MD simulations also show that the water density inside the UCP2 monomer is asymmetric, with the cytoplasmic side having a higher water density and a wider radius. In contrast, the structurally comparable adenosine 5'-diphosphate (ADP)/adenosine 5'-triphosphate (ATP) carrier (AAC1) did not form tetramers, implying that tetramerization cannot be generalized to all mitochondrial carriers.