Surface Mapping and Functional Interactions of GPx4, a Cytoprotective Peripheral Membrane Protein

Abstract

The study of peripheral membrane proteins (PMPs) has long been fraught with difficulty due to the need for reliable and robust membrane models. Many PMPs are soluble in an inactive state and recruited to the membrane to perform their respective function. This is the source of difficulty: the small size of the proteins and their inherent dynamic interactions with membranes and specific lipids make them incredibly challenging to characterize using X‐ray crystallography or cryo‐EM. Using protein NMR and better optimized membrane mimics, a robust study of PMPs may proceed.Glutathione peroxidase 4 (GPx4) is a PMP with the unique function of protection of membranes from lipid hydroperoxidation. This enzyme prevents cell death from ferroptosis by engaging the membrane and reducing lipid hydroperoxides. GPx4 has emerged as a high‐profile therapeutic target due to the ability of inhibitors to leverage ferroptosis against certain types of cancer cells.We utilize a wide variety of membrane models and native substrates to map the binding interaction of the GPx4 with the membrane and substrate lipids. Electrostatic interactions of the cationic patch are also found to be important in the moonlighting function of GPx4 as a cross‐linker of DNA‐bound protamines. Finally, our experimentally determined membrane interface using DPC micelles to formulate and confirm a novel DLPC:DPC reverse micelle system that can act as a membrane model for future PMP studies. The novel DLPC:DPC formulation is the first step in developing reverse micelles as more biologically accurate membrane models that can be tuned depending on membrane chemistry.