Cryo-EM structures of human STEAP4 reveal mechanism of iron(III) reduction

Abstract

Enzymes of the six-transmembrane epithelial antigen of the prostate (STEAP) family reduce Fe3+ and Cu2+ ions to facilitate metal-ion uptake by mammalian cells. STEAPs are highly upregulated in several types of cancer, making them potential therapeutic targets. However, the structural basis for STEAP-catalyzed electron transfer through an array of cofactors to metals at the membrane luminal side remains elusive. Here, we report cryo-electron microscopy structures of human STEAP4 in absence and presence of Fe3+-NTA. Domain-swapped, trimeric STEAP4 orients NADPH bound to a cytosolic domain onto axially aligned flavin-adenine dinucleotide (FAD) and a single b-type heme that cross the transmembrane-domain to enable electron transfer. Substrate binding within a positively charged ring indicates that iron gets reduced while in complex with its chelator. These molecular principles of iron reduction provide a basis for exploring STEAPs as therapeutic targets.

Structural basis for targeting human cancer antigen STEAP1 with antibodies

“…In our previous work, we showed that substrate iron(III), complexed to a negatively-charged chelator like citrate, binds in the basic ring of STEAP4 ( Fig. 4D) and we proposed that this ring of positive amino acids may polarize the iron(III)-chelator complex to facilitate the iron-reduction reaction (29). The presence of a comparable positively charged ring (Fig.…”

“…S3D), which is missing in STEAP1. In line with this, STEAP3 and 4 exhibit a low micromolar affinity for FAD (Kd = ~1 µM) (26,29), whereas the affinity of STEAP1 for FAD is much weaker (Kd = 34 µM) (30). The cryo-EM density in this region could therefore correspond to a loosely bound FAD cofactor, although the weak density does not allow for modelling the complete cofactor.…”

“…The protein production protocol was adapted from the previously described protocol for STEAP4 (29). Full-length STEAP1 with a C-terminal Strep3 tag was expressed in HEK293 GNTIsuspension cells (provided by U-Protein Express BV).…”

“…STEAP2-4 additionally contain an intracellular oxidoreductase domain (OxRD) that binds nicotinamide-adenine dinucleotide phosphate (NADPH) (27,28). The ferric and cupric reductase mechanism of STEAP2-4 is defined by electron transfer from intracellular NADPH through membrane-embedded flavin-adenine dinucleotide (FAD) and heme cofactors to chelated metal-ion complexes at the membrane extracellular side (26,29). Contrary to STEAP2-4, STEAP1 does not exhibit metalloreductase activity when over expressed on mammalian cells (19), suggesting that it may have a distinct, yet unidentified function.…”

“…In support, its expression often correlates with the expression of STEAP2 in cancers (17) and both proteins co-purify in detergent (30), suggesting they could form a functional complex. Further indications for a functional hetero-trimeric STEAP complex emerged from the recent cryo-EM structures of homo-trimeric human STEAP4 (29), which revealed a domain-swapped architecture, with the intracellular OxRD positioned beneath the TMD of the adjacent protomer. This arrangement supports a model in which the heme in STEAP1 receives electrons from NADPH bound to an adjacent STEAP2/3/4 subunit.…”