The structure of erastin-bound xCT–4F2hc complex reveals molecular mechanisms underlying erastin-induced ferroptosis

Ferroptosis is an iron-dependent, non-apoptotic form of regulated cell death characterized by an accumulation of lipid- derived reactive oxygen species (ROS). The small-molecule compound erastin induces ferroptosis via inhibiting the cystine-glutamate antiporter system x c – , which consists of two subunits, namely the light chain xCT and the heavy chain 4F2hc (encoded by the SLC7A11 and SLC3A2 genes, respectively). 1 , 2 , 3 , 4 Recent studies have shown that xCT (SLC7A11) regulates ferroptosis in liver fibrosis, cardiomyopathy, and numerous other pathophysiological processes. 5 , 6 , 7 , 8 The complex formed by xCT and 4F2hc has also been suggested as a possible therapeutic target for cancer, as it is overexpressed in a wide variety of cancer types; moreover, inhibiting xCT impairs cystine uptake, causing an accumulation of ROS and suppressing tumor growth. 9 , 10 However, the underlying molecular mechanisms remain unknown. Here, we overexpressed and then co-purified human xCT and 4F2hc, finding that they form a stable complex (Fig. 1a ). To test whether this purified complex is functional, we then reconstituted the complex into liposomes and performed a counter-flow assay. We found that the wild-type (WT) xCT–4F2hc complex mediates the exchange of cystine and glutamate — measured as the uptake of 14 C-labeled cystine — and this activity was significantly reduced by the system x c – inhibitors, erastin and sulfasalazine ( Supplementary information , Fig. S1 ). Fig. 1: Cryo-EM structure of the erastin-bound xCT–4F2hc complex and the inhibitory effect of erastin on xCT activity in ferroptosis. a Representative gel filtration purification of the xCT–4F2hc complex. The inset shows a Coomassie blue-stained SDS-PAGE gel under reducing (+DTT) and oxidizing (–DTT) conditions. M, molecular weight marker. The bands corresponding to 4F2hc, xCT, and the disulfide-linked xCT–4F2hc complex are labeled with a triangle, circle, and square, respectively. b , c Cryo-EM map ( b ) and atomic model ( c ) of the erastin-bound xCT–4F2hc complex. d Interaction interfaces between xCT and 4F2hc. The boxed regions (from top to bottom) are shown in e , f and g . e Detailed view of the interactions at the extracellular interface, with the polar interactions shown as red dashed lines. f Detailed view of the hydrophobic interactions between the transmembrane helix in 4F2hc and TM4 in xCT. g Detailed view of the xCT–4F2hc complex at the intracellular side; xCT is bound to two lipid molecules. h Structure of the erastin-bound xCT subunit, with the density for erastin shown in mesh. i Model of the erastin-binding pocket located in the intracellular vestibule of xCT. j The erastin molecule is shown sandwiched between TM domains in xCT; the tail of the erastin molecule is surrounded by a cluster of hydrophobic residues (shown as spheres). k Overlay showing the structure of the TM domains in erastin-bound xCT and erastin-bound LAT1 (PDB ID: 6IRT). l , m Dose-response curves for erastin ( l ) and IKE ( m ) measured in xCT-knockout HT-1080 cells expressing either WT xCT or the indicated xCT mutants; the corresponding IC 50 values are also shown. n Erastin-induced lipid peroxidation was measured using C11-BODIPY in xCT-knockout HT-1080 cells expressing either WT xCT or the indicated xCT mutants, 12 h after treatment with 2 μM erastin or DMSO as a negative control. The corresponding percentage of C11-BODIPY-positive values are shown. o Na + -independent [ 14 C]-cystine uptake was measured in xCT-knockout HT-1080 cells and in cells expressing either WT xCT or the indicated xCT mutants; where indicated, the cells were pretreated for 2 h with either 4 μM erastin or DMSO. In l , m *** P < 0.001 vs WT (one-way ANOVA); in o , * P < 0.05, *** P < 0.001, and n.s., not significant ( P > 0.05) (Student’s t -test). Full size image