Structure of the membrane proximal oxidoreductase domain of human Steap3, the dominant ferrireductase of the erythroid transferrin cycle Anoop K. Sendamarai*, Robert S. Ohgami†, Mark D. Fleming†, and C. Martin Lawrence*‡ *Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717; and †Department of Pathology, Children’s Hospital and Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115 Edited by Pamela J. Bjorkman, California Institute of Technology, Pasadena, CA, and approved March 20, 2008 (received for review February 8, 2008) The daily production of 200 billion erythrocytes requires 20 mg of undergo phagocytosis by macrophages, and much of the eryth- iron, accounting for nearly 80% of the iron demand in humans. rocyte iron is recycled, drastically reducing the need for dietary Thus, erythroid precursor cells possess an efficient mechanism for uptake of additional iron (9). To meet their iron need, erythroid iron uptake in which iron loaded transferrin (Tf) binds to the precursor cells are uniquely dependent on the transferrin cycle transferrin receptor (TfR) at the cell surface. The Tf:TfR complex (4, 10). In this cycle, ferric (Fe3ϩ) iron-loaded transferrin (Tf) then enters the endosome via receptor-mediated endocytosis. binds to the transferrin receptor (TfR-1) on the cell surface. The Upon endosomal acidification, iron is released from Tf, reduced to Tf:TfR-1 complex then enters the endosome via receptor- Fe2؉ by Steap3, and transported across the endosomal membrane mediated endocytosis. Within the endosome, iron is released by divalent metal iron transporter 1. Steap3, the major ferrireduc- from Tf and then is reduced from Fe3ϩ to Fe2ϩ by Steap3, tase in erythrocyte endosomes, is a member of a unique family of permitting transport across the endosomal membrane by diva- reductases. Steap3 is comprised of an N-terminal cytosolic oxi- lent metal iron transporter 1 (DMT1), which is selective for doreductase domain and a C-terminal heme-containing transmem- Fe2ϩ. The apo-Tf:TfR-1 complex is then recycled to the cell brane domain. Cytosolic NADPH and a flavin are predicted cofac- surface, where, at neutral pH, the apo-Tf is released to partic- tors, but the NADPH/flavin binding domain differs significantly ipate in the cycle once again (4, 10). from those in other eukaryotic reductases. Instead, Steap3 shows Ohgami et al. (11, 12) recently demonstrated that Steap3, a remarkable, although limited homology to FNO, an archaeal oxi- member of a unique family of transmembrane reductases, is the doreductase. We have determined the crystal structure of the major erythroid ferrireductase. Steap (six transmembrane epi- human Steap3 oxidoreductase domain in the absence and presence thelial antigen of the prostate) family members contain a of NADPH. The structure reveals an FNO-like domain with an C-terminal domain composed of six transmembrane helices and unexpected dimer interface and substrate binding sites that are are thought to coordinate a single intramembrane heme via two well positioned to direct electron transfer from the cytosol to a conserved histidine residues (12–14). With the exception of heme moiety predicted to be fixed within the transmembrane Steap1, the transmembrane domain is accompanied by an N- domain. Here, we discuss possible gating mechanisms for electron terminal oxidoreductase domain predicted to lie against the transfer across the endosomal membrane. cytosolic face of the membrane (13). Accordingly, Steap2 and Steap4 also exhibit ferrireductase activity in vitro and, along with iron transport ͉ ferric ͉ erythrocyte Steap3, have been shown to stimulate uptake of non-transferrin- bound iron and copper (13). These activities strongly suggest a ron plays an integral role in many biochemical processes greater role for Steap proteins in iron and copper metabolism. Iessential to life. However, unsequestered Fe2ϩ is deleterious, Not surprisingly then, Steap proteins appear to play important because it catalyzes production of the hydroxyl free radical via roles in human health. Steap1 is expressed in many human cancer the Fenton reaction (1). For these reasons, iron homeostasis is cell lines (15) and, with Steap2, is found at particularly high levels critical in human health, and diseases of iron transport and in prostate cancer (16), making Steap1 an appealing target for metabolism are among the most prevalent causes of morbidity in cancer immunotherapy (15–19). Mice lacking Steap3 exhibit Ϫ/Ϫ humans (2). Iron deficiency is thought to affect more than one hypochromic microcytic anemia (11, 12). Although Steap4 billion people worldwide, particularly pregnant women and knockout mice develop spontaneous metabolic disease on a young children (3, 4) and is largely due to dietary insufficiency regular diet, manifesting insulin resistance, glucose intolerance, or excess loss. In contrast, iron overload disorders, collectively mild hyperglycemia, dyslipidemia, and fatty liver disease (20), known as hereditary hemochromatosis, are among the most reminiscent of ‘‘metabolic syndrome’’ in humans (20–22). frequent single gene disorders in humans (5, 6). The occurrence The Steap family oxidoreductase domain differs significantly of a single disease associated allele, HFEC282Y, is as high as 10% from those of the bacterial Fre, yeast FRE, or plant FRO in individuals of Northern European descent (7). In homozygous families of transmembrane metalloreductases. In particular, individuals, progressive iron accumulation generates oxidative stress that results in significant cellular damage, with induction Author contributions: M.D.F. and C.M.L. designed research; A.K.S. and R.S.O. performed of inflammation and fibrosis that eventuates in hepatic cirrhosis, research; R.S.O. contributed new reagents/analytic tools; A.K.S. and C.M.L. analyzed data; hepatocellular carcinoma, diabetes mellitus, cardiac insuffi- and A.K.S., R.S.O., M.D.F., and C.M.L. wrote the paper. ciency, and arthropathy (8). Consequently, the cellular machin- The authors declare no conflict of interest. ery and mechanisms responsible for iron transport and ho- This article is a PNAS Direct Submission. meostasis are worthy of significant investigation, because they Data deposition: The coordinates and structure factors have been deposited in the Protein may provide targets for pharmacological intervention to either Data Bank, www.pdb.org (PDB ID codes 2VNS and 2VQ3). promote or inhibit cellular iron uptake in a variety of human ‡To whom correspondence should be addressed. E-mail: [email protected]. disorders. edu. The daily production of 200 billion erythrocytes requires Ϸ20 This article contains supporting information online at www.pnas.org/cgi/content/full/ mg of iron, accounting for nearly 80% of the iron demand in 0801318105/DCSupplemental. humans (4). However, as the red blood cells senesce, they © 2008 by The National Academy of Sciences of the USA 7410–7415 ͉ PNAS ͉ May 27, 2008 ͉ vol. 105 ͉ no. 21 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0801318105 Downloaded by guest on September 26, 2021 Steap3 contains an unusual oxidoreductase domain that shows The structure of the subunit appears as a fusion of two ϩ limited similarity (28% identity) to F420H2:NADP oxidoreduc- subdomains (Fig. 1B). At the N-terminal end, residues 29–146 tase (FNO) from Archaeoglobus fulgidus (23). This archaeal form a classical nucleotide binding domain composed of a enzyme utilizes F420, a unique 5Ј deazaflavin derivative that is six-stranded parallel ␤-sheet and five flanking ␣-helices (␣1–␣4 unknown in mammals. Thus, the FNO-like domain in the and ␣6). However, strand ␤5 extends significantly beyond the mammalian Steap family is likely to use a more commonly found neighboring strands, where it enters an unanticipated ␣-helix flavin derivative such as FMN or FAD for this purpose. The (␣5) before returning to helix ␣6 and strand ␤6, canonical reduction of iron is thought to occur by the sequential transfer elements of the nucleotide binding domain. Residues 147–208 of electrons from cytosolic NADPH to endosomal Fe3ϩ via the then form a smaller C-terminal subdomain that begins with a flavin derivative and the intramembrane heme (11–13). Impor- short ␣-helix (␣7) that emanates from ␤6 of the nucleotide tantly, the Steap3 oxidoreducatase domain is not present in binding domain. The ␣7 helix is followed by a poorly ordered yeast, nematodes or fruit flies, and in mammals is found only in loop (Gly157-Asn162) that then connects to a ␤-␣-␤ supersecond- other Steap family members, specifically Steap2 and Steap4. For ary structural element (␤7-␣8-␤8) that joins the nucleotide this reason, the FNO-like domains of these proteins may rep- binding domain to form a twisted, 8-stranded ␤-sheet. Although resent excellent targets for pharmacological intervention (11, strands ␤7 and ␤8 run parallel to each other, they lie antiparallel 12, 13). to those in the nucleotide binding domain. A final ␣-helix (␣9) then runs back across the top edge of the ␤-sheet, followed by Results a few C-terminal residues that approach the dimer axis. As Biochemical Characterization. The human Steap3 construct used in expected, significant similarities between Steap3 and the ar- this study includes an N-terminal His-tag and residues 1–215 of chaeal FNO are found. The Steap3 core superposes on FNO the cytoplasmic oxidoreductase domain. Interestingly, we ob- (PDB entry 1JAX) with an rmsd of 1.44 Å [162 structurally serve two species of purified Steap3 oxidoreductase by size equivalent residues,