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Characterization of a Single b-type Heme, FAD, and Metal Binding Sites in the Transmembrane Domain of Six-transmembrane Epithelial Antigen of the Prostate (STEAP) Family Proteins*

  • Mark D. Kleven
    Affiliations
    Departments of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717
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  • Mensur Dlakić
    Affiliations
    Departments of Microbiology and Immunology, Montana State University, Bozeman, Montana 59717
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  • C. Martin Lawrence
    Correspondence
    To whom correspondence may be addressed. Tel.: 406-994-5382; Fax: 406-994-5407.
    Affiliations
    Departments of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant RO1 GM084326 (to C. M. L. and M. D. F.). The authors declare that they have no conflicts of interest with the contents of this article.
    This article contains supplemental Fig. 1 and a list showing Steap family alignment species.
Open AccessPublished:July 23, 2015DOI:https://doi.org/10.1074/jbc.M115.664565
      Six-transmembrane epithelial antigen of the prostate 3 (Steap3) is the major ferric reductase in developing erythrocytes. Steap family proteins are defined by a shared transmembrane domain that in Steap3 has been shown to function as a transmembrane electron shuttle, moving cytoplasmic electrons derived from NADPH across the lipid bilayer to the extracellular face where they are used to reduce Fe3+ to Fe2+ and potentially Cu2+ to Cu1+. Although the cytoplasmic N-terminal oxidoreductase domain of Steap3 and Steap4 are relatively well characterized, little work has been done to characterize the transmembrane domain of any member of the Steap family. Here we identify high affinity FAD and iron biding sites and characterize a single b-type heme binding site in the Steap3 transmembrane domain. Furthermore, we show that Steap3 is functional as a homodimer and that it utilizes an intrasubunit electron transfer pathway through the single heme moiety rather than an intersubunit electron pathway through a potential domain-swapped dimer. Importantly, the sequence motifs in the transmembrane domain that are associated with the FAD and metal binding sites are not only present in Steap2 and Steap4 but also in Steap1, which lacks the N-terminal oxidoreductase domain. This strongly suggests that Steap1 harbors latent oxidoreductase activity.

      Introduction

      The daily production of 200 billion erythrocytes accounts for nearly 80% of the total iron demand in humans (
      • Hentze M.W.
      • Muckenthaler M.U.
      • Andrews N.C.
      Balancing acts: molecular control of mammalian iron metabolism.
      ). To meet this need, developing erythrocytes utilize the transferrin cycle to import iron into the cell. In this process, iron-loaded transferrin is bound at the cell surface by the transferrin receptor. This is followed by endocytosis and acidification of the endosomal compartment, which promotes release of Fe3+. The Fe3+ is then reduced to Fe2+ by six-transmembrane epithelial antigen of the prostate 3 (Steap3),
      The abbreviations used are: Steap
      six-transmembrane antigen of the prostate
      ALA
      δ-aminolevulinic acid
      BiFC
      bimolecular fluorescence complementation
      F420
      8-hydroxy-5-deazaflavin
      FRD
      ferric reductase domain
      NTA
      nitrilotriacetic acid.
      the major ferric reductase of the erythroid transferrin cycle (
      • Ohgami R.S.
      • Campagna D.R.
      • Greer E.L.
      • Antiochos B.
      • McDonald A.
      • Chen J.
      • Sharp J.J.
      • Fujiwara Y.
      • Barker J.E.
      • Fleming M.D.
      Identification of a ferrireductase required for efficient transferrin-dependent iron uptake in erythroid cells.
      ,
      • Ohgami R.S.
      • Campagna D.R.
      • McDonald A.
      • Fleming M.D.
      The Steap proteins are metalloreductases.
      ). Finally, Fe2+ is transported across the endosomal membrane by divalent metal transporter 1 where it supports the synthesis of hemoglobin and other cellular needs or in iron-replete cells is sequestered within the iron storage protein ferritin.
      Among proteins comprising the transferrin cycle, Steap3 is the most recent to be identified (
      • Ohgami R.S.
      • Campagna D.R.
      • Greer E.L.
      • Antiochos B.
      • McDonald A.
      • Chen J.
      • Sharp J.J.
      • Fujiwara Y.
      • Barker J.E.
      • Fleming M.D.
      Identification of a ferrireductase required for efficient transferrin-dependent iron uptake in erythroid cells.
      ,
      • Ohgami R.S.
      • Campagna D.R.
      • McDonald A.
      • Fleming M.D.
      The Steap proteins are metalloreductases.
      ,
      • Ohgami R.S.
      • Campagna D.R.
      • Antiochos B.
      • Wood E.B.
      • Sharp J.J.
      • Barker J.E.
      • Fleming M.D.
      nm1054: a spontaneous, recessive, hypochromic, microcytic anemia mutation in the mouse.
      ). The work by Ohgami et al. (
      • Ohgami R.S.
      • Campagna D.R.
      • Greer E.L.
      • Antiochos B.
      • McDonald A.
      • Chen J.
      • Sharp J.J.
      • Fujiwara Y.
      • Barker J.E.
      • Fleming M.D.
      Identification of a ferrireductase required for efficient transferrin-dependent iron uptake in erythroid cells.
      ) included protein homology analyses that provided initial clues to its function and mechanism. Steap3 and its homologs Steap2 and Steap4 were predicted to be composed of two distinct domains: an N-terminal cytoplasmic domain and a C-terminal transmembrane domain (
      • Ohgami R.S.
      • Campagna D.R.
      • Greer E.L.
      • Antiochos B.
      • McDonald A.
      • Chen J.
      • Sharp J.J.
      • Fujiwara Y.
      • Barker J.E.
      • Fleming M.D.
      Identification of a ferrireductase required for efficient transferrin-dependent iron uptake in erythroid cells.
      ,
      • Hubert R.S.
      • Vivanco I.
      • Chen E.
      • Rastegar S.
      • Leong K.
      • Mitchell S.C.
      • Madraswala R.
      • Zhou Y.
      • Kuo J.
      • Raitano A.B.
      • Jakobovits A.
      • Saffran D.C.
      • Afar D.E.
      STEAP: a prostate-specific cell-surface antigen highly expressed in human prostate tumors.
      ,
      • Moldes M.
      • Lasnier F.
      • Gauthereau X.
      • Klein C.
      • Pairault J.
      • Fève B.
      • Chambaut-Guérin A.M.
      Tumor necrosis factor-α-induced adipose-related protein (TIARP), a cell-surface protein that is highly induced by tumor necrosis factor-α and adipose conversion.
      ,
      • Porkka K.P.
      • Nupponen N.N.
      • Tammela T.L.
      • Vessella R.L.
      • Visakorpi T.
      Human pHyde is not a classical tumor suppressor gene in prostate cancer.
      ). The closest homolog for the N-terminal cytoplasmic domain is the prokaryotic F420:NADP+ oxidoreductase. F420H2:NADP+ oxidoreductase utilizes an elaborated Rossmann or dinucleotide binding domain to bind NADPH and the flavin-derivative F420 (
      • Warkentin E.
      • Mamat B.
      • Sordel-Klippert M.
      • Wicke M.
      • Thauer R.K.
      • Iwata M.
      • Iwata S.
      • Ermler U.
      • Shima S.
      Structures of F420H2:NADP+ oxidoreductase with and without its substrates bound.
      ). In methanogens, F420H2:NADP+ oxidoreductase reduces F420 to F420H2, which is subsequently used to reduce CO2 to methane. Because the flavin analog F420 is not known in mammals, the homology suggested that the N-terminal domain of Steap3 would instead bind NAD(P)H and a flavin, such as FAD or FMN.
      The C-terminal domain was predicted to contain six transmembrane α-helices with distant homology to yeast ferric reductases and to mammalian NADPH oxidase among others (
      • Ohgami R.S.
      • Campagna D.R.
      • Greer E.L.
      • Antiochos B.
      • McDonald A.
      • Chen J.
      • Sharp J.J.
      • Fujiwara Y.
      • Barker J.E.
      • Fleming M.D.
      Identification of a ferrireductase required for efficient transferrin-dependent iron uptake in erythroid cells.
      ). Thus, collectively, the transmembrane domains of the Steap, ferric reductase, and NADPH oxidase protein families place each of these within the greater “ferric reductase domain” (FRD) superfamily (
      • Zhang X.
      • Krause K.H.
      • Xenarios I.
      • Soldati T.
      • Boeckmann B.
      Evolution of the ferric reductase domain (FRD) superfamily: modularity, functional diversification, and signature motifs.
      ). In this light, members of the ferric reductase and NADPH oxidase families utilize cytosolic domains or subunits to transfer electrons from NADPH to FAD. The reduced FADH2 then transfers its electrons to the transmembrane domain of these proteins, which utilizes two embedded heme cofactors to move the electrons across the membrane to the extracellular or luminal face of the protein for the reduction of iron or O2, respectively. For ferric reductase and NADPH oxidase, the two heme moieties are coordinated by four histidine residues in their transmembrane segments. Interestingly, in Steap3 and its mammalian homologs, only two of these histidine residues are conserved, thus predicting only a single transmembrane heme (
      • Ohgami R.S.
      • Campagna D.R.
      • Greer E.L.
      • Antiochos B.
      • McDonald A.
      • Chen J.
      • Sharp J.J.
      • Fujiwara Y.
      • Barker J.E.
      • Fleming M.D.
      Identification of a ferrireductase required for efficient transferrin-dependent iron uptake in erythroid cells.
      ).
      The crystal structures of the truncated cytoplasmic oxidoreductase domains have been determined for Steap3 and Steap4 (
      • Sendamarai A.K.
      • Ohgami R.S.
      • Fleming M.D.
      • Lawrence C.M.
      Structure of the membrane proximal oxidoreductase domain of human Steap3, the dominant ferrireductase of the erythroid transferrin cycle.
      ,
      • Gauss G.H.
      • Kleven M.D.
      • Sendamarai A.K.
      • Fleming M.D.
      • Lawrence C.M.
      The crystal structure of six-transmembrane epithelial antigen of the prostate 4 (Steap4), a ferri/cuprireductase, suggests a novel interdomain flavin-binding site.
      ). Their structures are quite similar with a backbone root mean square deviation of 1 Å (
      • Gauss G.H.
      • Kleven M.D.
      • Sendamarai A.K.
      • Fleming M.D.
      • Lawrence C.M.
      The crystal structure of six-transmembrane epithelial antigen of the prostate 4 (Steap4), a ferri/cuprireductase, suggests a novel interdomain flavin-binding site.
      ). In each case, these oxidoreductase domains crystallized as 2-fold symmetric dimers. Furthermore, the N-terminal Steap3 oxidoreductase domain was found to dimerize at low millimolar concentrations in solution. Collectively, these observations suggest that full-length Steap3 may be present in cellular membranes as a homodimer.
      As expected, F420H2:NADP+ oxidoreductase was indeed found to be a structural homolog to the Steap oxidoreductase domain (Steap3 backbone root mean square deviation of 1.44 Å) (
      • Sendamarai A.K.
      • Ohgami R.S.
      • Fleming M.D.
      • Lawrence C.M.
      Structure of the membrane proximal oxidoreductase domain of human Steap3, the dominant ferrireductase of the erythroid transferrin cycle.
      ). Furthermore, like F420H2:NADP+ oxidoreductase, both Steap3 and Steap4 were co-crystallized with NADPH. Unlike F420H2:NADP+ oxidoreductase, however, neither could be co-crystallized with a flavin. And in contrast to F420H2:NADP+ oxidoreductase, the crystal structures of the isolated oxidoreductase domains of Steap3 and Steap4 show that the nicotinamide ring of NADPH lies in an open, solvent-exposed pocket.
      Although structural studies of the isolated Steap3 and Steap4 oxidoreductase domains failed to identify a flavin binding site, flavin-dependent NADPH oxidase activity was observed for the truncated Steap4 domain (
      • Gauss G.H.
      • Kleven M.D.
      • Sendamarai A.K.
      • Fleming M.D.
      • Lawrence C.M.
      The crystal structure of six-transmembrane epithelial antigen of the prostate 4 (Steap4), a ferri/cuprireductase, suggests a novel interdomain flavin-binding site.
      ). However, this truncated construct exhibits extremely high, nonphysiological Km values (>100 μm) for each of the flavins examined (FAD, FMN, and riboflavin). Thus, contrary to initial expectations, these observations collectively suggested that the oxidoreductase domain does not contain a fully functional flavin binding site. Furthermore, by process of elimination, it also suggested that structural elements within the C-terminal transmembrane domain could play a major role in flavin recognition.
      Thus, although biochemical and structural studies have provided valuable insights into Steap structure-function relationships in the N-terminal oxidoreductase domain, many questions remain regarding the C-terminal transmembrane domain and the full-length protein. Specifically, although sequence analysis identifies one pair of heme-coordinating residues, does the transmembrane domain indeed contain only a single heme cofactor? Or is there a second, cryptic heme present; and if so, how is it bound, and what type of heme is utilized (heme a, b, or c)? Does the full-length protein form a homodimer, and if so, what is the functional impact of dimerization? Does the full-length protein bind a specific flavin with high affinity? Does the transmembrane domain play a pivotal role in flavin recognition, and if so, which elements in the transmembrane domain are responsible for high affinity flavin recognition? Finally, where is the iron binding site? Does the iron bind adjacent to the heme, or are electrons transferred from the heme to an iron bound at a more distant site?

      Author Contributions

      M. D. K. was responsible for the bulk of the data acquisition. M. D. made the Gateway vectors and helped revise the manuscript. M. D. K. and C. M. L. conceived and coordinated the study and wrote and revised the paper.

      Acknowledgments

      We are deeply grateful to Mark D. Fleming for valuable insight and careful reading of the manuscript. We thank Chang-Deng Hu (Purdue University) for providing Venus plasmids that were used for making Gateway vectors. The Montana State University confocal microscope was purchased with funding from the National Science Foundation Major Research Instrumentation Program and the M. J. Murdock Charitable Trust. We also thank Jan Phillipp Kreysing for gracious help in producing Fig. 6.

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