BPS2026 – Experimental and computational investigation of the mechanism of chloride dependence in system xC-

The human transmembrane protein system x C - , an antiporter of glutamate and cystine, has been targeted in the treatment of cancer and a wide array of neurodegenerative disorders due to its roles in combating ferroptosis and oxidative stress. Analysis of system x C - activity has shown cystine uptake to be dependent on extracellular chloride concentration. The objective of our study is to understand this chloride dependence. We have used all-atom molecular dynamics (MD) simulations to guide site-directed mutagenesis. In a series of MD simulations, one of two versions of the system x C - protein (full or truncated) was embedded in a bilayer of 5:1 POPC:SAPE and solvated with OPC waters and one of four concentrations of NaCl (0, 100, 300, or 1000 mM). By analyzing chloride density from all of these simulations (over 50 μs total), three likely chloride binding sites were identified in apo system x C - . From these three sites, nine residues of interest were identified and nine system x C - mutants have been created: H110A, R126A, E130A, R135A, Q191A, K198A, Y244F, F250A, Y251F, S330A, and R396A. Preliminary analysis of transport function in these mutants and wild type system x C - will also be presented.