Cryo-electron microscopy structure and potential enzymatic function of human six-transmembrane epithelial antigen of the prostate 1 (STEAP1)
2020; Elsevier BV; Volume: 295; Issue: 28 Linguagem: Inglês
10.1074/jbc.ra120.013690
ISSN1083-351X
Autores Tópico(s)Electron and X-Ray Spectroscopy Techniques
ResumoSix-transmembrane epithelial antigen of the prostate 1 (STEAP1) is an integral membrane protein that is highly up-regulated on the cell surface of several human cancers, making it a promising therapeutic target to manage these diseases. It shares sequence homology with three enzymes (STEAP2–STEAP4) that catalyze the NADPH-dependent reduction of iron(III). However, STEAP1 lacks an intracellular NADPH-binding domain and does not exhibit cellular ferric reductase activity. Thus, both the molecular function of STEAP1 and its role in cancer progression remain elusive. Here, we present a ∼3.0-Å cryo-EM structure of trimeric human STEAP1 bound to three antigen-binding fragments (Fabs) of the clinically used antibody mAb120.545. The structure revealed that STEAP1 adopts a reductase-like conformation and interacts with the Fabs through its extracellular helices. Enzymatic assays in human cells revealed that STEAP1 promotes iron(III) reduction when fused to the intracellular NADPH-binding domain of its family member STEAP4, suggesting that STEAP1 functions as a ferric reductase in STEAP heterotrimers. Our work provides a foundation for deciphering the molecular mechanisms of STEAP1 and may be useful in the design of new therapeutic strategies to target STEAP1 in cancer. Six-transmembrane epithelial antigen of the prostate 1 (STEAP1) is an integral membrane protein that is highly up-regulated on the cell surface of several human cancers, making it a promising therapeutic target to manage these diseases. It shares sequence homology with three enzymes (STEAP2–STEAP4) that catalyze the NADPH-dependent reduction of iron(III). However, STEAP1 lacks an intracellular NADPH-binding domain and does not exhibit cellular ferric reductase activity. Thus, both the molecular function of STEAP1 and its role in cancer progression remain elusive. Here, we present a ∼3.0-Å cryo-EM structure of trimeric human STEAP1 bound to three antigen-binding fragments (Fabs) of the clinically used antibody mAb120.545. The structure revealed that STEAP1 adopts a reductase-like conformation and interacts with the Fabs through its extracellular helices. Enzymatic assays in human cells revealed that STEAP1 promotes iron(III) reduction when fused to the intracellular NADPH-binding domain of its family member STEAP4, suggesting that STEAP1 functions as a ferric reductase in STEAP heterotrimers. Our work provides a foundation for deciphering the molecular mechanisms of STEAP1 and may be useful in the design of new therapeutic strategies to target STEAP1 in cancer. Since its discovery in 1999 as a multispan membrane protein highly expressed on prostate cancer cells (1Hubert 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.Proc. Natl. Acad. Sci. U.S.A. 1999; 96 (10588738): 14523-1452810.1073/pnas.96.25.14523Crossref PubMed Scopus (269) Google Scholar), six-transmembrane epithelial antigen of the prostate 1 (STEAP1) emerged as a cancer antigen expressed in various human cancers, including prostate, bladder, colorectal, lung, ovarian, and breast carcinoma and Ewing sarcoma. Because its expression in physiological tissues is minimal and mainly confined to the prostate gland (2Gomes I.M. Maia C.J. Santos C.R. STEAP proteins: from structure to applications in cancer therapy.Mol. Cancer Res. 2012; 10 (22522456): 573-58710.1158/1541-7786.MCR-11-0281Crossref PubMed Scopus (117) Google Scholar), STEAP1 represents a potentially attractive therapeutic tool as both a cancer biomarker and a target for anticancer therapies (2Gomes I.M. Maia C.J. Santos C.R. STEAP proteins: from structure to applications in cancer therapy.Mol. Cancer Res. 2012; 10 (22522456): 573-58710.1158/1541-7786.MCR-11-0281Crossref PubMed Scopus (117) Google Scholar, 3Moreaux J. Kassambara A. Hose D. Klein B. STEAP1 is overexpressed in cancers: a promising therapeutic target.Biochem. Biophys. Res. Commun. 2012; 429 (23142226): 148-15510.1016/j.bbrc.2012.10.123Crossref PubMed Scopus (53) Google Scholar, 4Barroca-Ferreira J. Pais J.P. Santos M.M. Goncalves A.M. Gomes I.M. Sousa I. Rocha S.M. Passarinha L.A. Maia C.J. Targeting STEAP1 protein in human cancer: current trends and future challenges.Curr. Cancer Drug Targets. 2018; 18 (28460619): 222-23010.2174/1568009617666170427103732Crossref PubMed Scopus (27) Google Scholar). Indeed, several strategies for targeting STEAP1 in cancer have been explored; in 2007, a study reported the production and characterization of two monoclonal antibodies (mAb120.545 and mAb92.30) that bind STEAP1 with nanomolar affinity on prostate cancer cells and inhibit the growth of prostate and bladder tumor xenografts in mice (5Challita-Eid P.M. Morrison K. Etessami S. An Z. Morrison K.J. Perez-Villar J.J. Raitano A.B. Jia X.C. Gudas J.M. Kanner S.B. Jakobovits A. Monoclonal antibodies to six-transmembrane epithelial antigen of the prostate-1 inhibit intercellular communication in vitro and growth of human tumor xenografts in vivo.Cancer Res. 2007; 67 (17575147): 5798-580510.1158/0008-5472.CAN-06-3849Crossref PubMed Scopus (73) Google Scholar). More recently, clinical studies employing humanized variants of mAb120.545 that target STEAP1 were conducted, including 1) a phase I trial using an antibody-drug conjugate (termed DSTP3086S or Vandortuzumab Vedotin) to target prostate cancer (6Boswell C.A. Mundo E.E. Zhang C. Bumbaca D. Valle N.R. Kozak K.R. Fourie A. Chuh J. Koppada N. Saad O. Gill H. Shen B.Q. Rubinfeld B. Tibbitts J. Kaur S. et al.Impact of drug conjugation on pharmacokinetics and tissue distribution of anti-STEAP1 antibody-drug conjugates in rats.Bioconjug. Chem. 2011; 22 (21913715): 1994-200410.1021/bc200212aCrossref PubMed Scopus (162) Google Scholar, 7Sukumaran S. Zhang C. Leipold D.D. Saad O.M. Xu K. Gadkar K. Samineni D. Wang B. Milojic-Blair M. Carrasco-Triguero M. Rubinfeld B. Fielder P. Lin K. Ramanujan S. Development and translational application of an integrated, mechanistic model of antibody-drug conjugate pharmacokinetics.AAPS J. 2017; 19 (27679517): 130-14010.1208/s12248-016-9993-zCrossref PubMed Scopus (13) Google Scholar, 8Danila D.C. Szmulewitz R.Z. Vaishampayan U. Higano C.S. Baron A.D. Gilbert H.N. Brunstein F. Milojic-Blair M. Wang B. Kabbarah O. Mamounas M. Fine B.M. Maslyar D.J. Ungewickell A. Scher H.I. Phase I study of DSTP3086S, an antibody-drug conjugate targeting six-transmembrane epithelial antigen of prostate 1, in metastatic castration-resistant prostate cancer.J. Clin. Oncol. 2019; 37 (31689155): 3518-352710.1200/JCO.19.00646Crossref PubMed Scopus (27) Google Scholar) and 2) a combined phase I/phase II trial for the PET imaging of metastatic castration-resistant prostate cancer using Zr89-labelled antibody (termed [89Zr]Zr-DFO-MSTP2109A) (9Doran M.G. Watson P.A. Cheal S.M. Spratt D.E. Wongvipat J. Steckler J.M. Carrasquillo J.A. Evans M.J. Lewis J.S. Annotating STEAP1 regulation in prostate cancer with 89Zr immuno-PET.J. Nucl. Med. 2014; 55 (25453051): 2045-204910.2967/jnumed.114.145185Crossref PubMed Scopus (23) Google Scholar, 10O'Donoghue J.A. Danila D.C. Pandit-Taskar N. Beylergil V. Cheal S.M. Fleming S.E. Fox J.J. Ruan S. Zanzonico P.B. Ragupathi G. Lyashchenko S.K. Williams S.P. Scher H.I. Fine B.M. Humm J.L. et al.Pharmacokinetics and biodistribution of a [89Zr]Zr-DFO-MSTP2109A anti-STEAP1 antibody in metastatic castration-resistant prostate cancer patients.Mol. Pharmacol. 2019; 16 (31117485): 3083-309010.1021/acs.molpharmaceut.9b00326Crossref Scopus (20) Google Scholar, 11Carrasquillo J.A. Fine B. Pandit-Taskar N. Larson S.M. Fleming S. Fox J.J. Cheal S.M. O'Donoghue J.A. Ruan S. Ragupathi G. Lyashchenko S.K. Humm J.L. Scher H.I. Gonen M. Williams S. et al.Imaging patients with metastatic castration-resistant prostate cancer using 89Zr-DFO-MSTP2109A anti-STEAP1 antibody.J. Nucl. Med. 2019; 60: 1517-152310.2967/jnumed.118.222844Crossref PubMed Scopus (30) Google Scholar). Besides antibody-based strategies, several in vitro and in vivo studies revealed that STEAP1-derived peptides are immunogenic and thus suitable for recognition by cytotoxic T lymphocytes (12Alves P.M.S. Faure O. Graff-Dubois S. Cornet S. Bolonakis I. Gross D.A. Miconnet I. Chouaib S. Fizazi K. Soria J.C. Lemonnier F.A. Kosmatopoulos K. STEAP, a prostate tumor antigen, is a target of human CD8+ T cells.Cancer Immunol. Immunother. 2006; 55 (16622681): 1515-152310.1007/s00262-006-0165-3Crossref PubMed Scopus (56) Google Scholar, 13Schirmer D. Grünewald T.G.P. Klar R. Schmidt O. Wohlleber D. Rubío R.A. Uckert W. Thiel U. Bohne F. Busch D.H. Krackhardt A.M. Burdach S. Richter G.H.S. Transgenic antigen-specific, HLA-A*02:01-allo-restricted cytotoxic T cells recognize tumor-associated target antigen STEAP1 with high specificity.Oncoimmunology. 2016; 5: e1175795-1110.1080/2162402X.2016.1175795Crossref PubMed Scopus (15) Google Scholar, 14Herrmann V.L. Wieland D.E. Legler D.F. Wittmann V. Groettrup M. The STEAP1262-270 peptide encapsulated into PLGA microspheres elicits strong cytotoxic T cell immunity in HLA-A*0201 transgenic mice: a new approach to immunotherapy against prostate carcinoma.Prostate. 2016; 76 (26715028): 456-46810.1002/pros.23136Crossref PubMed Scopus (12) Google Scholar, 15Cappuccini F. Stribbling S. Pollock E. Hill A.V.S. Redchenko I. Immunogenicity and efficacy of the novel cancer vaccine based on simian adenovirus and MVA vectors alone and in combination with PD-1 mAb in a mouse model of prostate cancer.Cancer Immunol. Immunother. 2016; 65 (27052571): 701-71310.1007/s00262-016-1831-8Crossref PubMed Scopus (35) Google Scholar, 16Chen X. Wang R. Chen A. Wang Y. Wang Y. Zhou J. Cao R. Inhibition of mouse RM-1 prostate cancer and B16F10 melanoma by the fusion protein of HSP65 & STEAP1186-193.Biomed. Pharmacother. 2019; 111 (30841425): 1124-113110.1016/j.biopha.2019.01.012Crossref PubMed Scopus (11) Google Scholar), indicating that STEAP1 could represent a potential candidate for the development of anticancer vaccines (4Barroca-Ferreira J. Pais J.P. Santos M.M. Goncalves A.M. Gomes I.M. Sousa I. Rocha S.M. Passarinha L.A. Maia C.J. Targeting STEAP1 protein in human cancer: current trends and future challenges.Curr. Cancer Drug Targets. 2018; 18 (28460619): 222-23010.2174/1568009617666170427103732Crossref PubMed Scopus (27) Google Scholar, 17Grunewald T.G.P. Bach H. Cossarizza A. Matsumoto I. The STEAP protein family: versatile oxidoreductases and targets for cancer immunotherapy with overlapping and distinct cellular functions.Biol. Cell. 2012; 104 (22804687): 641-65710.1111/boc.201200027Crossref PubMed Scopus (62) Google Scholar). STEAP1 belongs to a protein family that comprises three metalloreductases (18Ohgami 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.Nat. Genet. 2005; 37 (16227996): 1264-126910.1038/ng1658Crossref PubMed Scopus (496) Google Scholar, 19Ohgami R.S. Campagna D.R. McDonald A. Fleming M.D. The Steap proteins are metalloreductases.Blood. 2006; 108 (16609065): 1388-139410.1182/blood-2006-02-003681Crossref PubMed Scopus (433) Google Scholar), STEAP2–STEAP4, also known as STAMP1–STAMP3 (20Korkmaz K.S. Elbi C. Korkmaz C.G. Loda M. Hager G.L. Saatcioglu F. Molecular cloning and characterization of STAMP1, a highly prostate-specific six transmembrane protein that is overexpressed in prostate cancer.J. Biol. Chem. 2002; 277 (12095985): 36689-3669610.1074/jbc.M202414200Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 21Korkmaz C.G. Korkmaz K.S. Kurys P. Elbi C. Wang L. Klokk T.I. Hammarstrom C. Troen G. Svindland A. Hager G.L. Saatcioglu F. Molecular cloning and characterization of STAMP2, an androgen-regulated six transmembrane protein that is overexpressed in prostate cancer.Oncogene. 2005; 24 (15897894): 4934-494510.1038/sj.onc.1208677Crossref PubMed Scopus (101) Google Scholar, 22Sikkeland J. Sheng X. Jin Y. Saatcioglu F. STAMPing at the crossroads of normal physiology and disease states.Mol. Cell. Endocrinol. 2016; 425 (26911931): 26-3610.1016/j.mce.2016.02.013Crossref PubMed Scopus (14) Google Scholar), which reduce iron(III) and copper(II) and are also associated with cancer progression (23Whiteland H. Spencer-Harty S. Morgan C. Kynaston H. Thomas D.H. Bose P. Fenn N. Lewis P. Jenkins S. Doak S.H. A role for STEAP2 in prostate cancer progression.Clin. Exp. Metastasis. 2014; 31 (25248617): 909-92010.1007/s10585-014-9679-9Crossref PubMed Scopus (35) Google Scholar, 24Isobe T. Baba E. Arita S. Komoda M. Tamura S. Shirakawa T. Ariyama H. Takaishi S. Kusaba H. Ueki T. Akashi K. Human STEAP3 maintains tumor growth under hypoferric condition.Exp. Cell Res. 2011; 317 (21871451): 2582-259110.1016/j.yexcr.2011.07.022Crossref PubMed Scopus (26) Google Scholar, 25Jin Y. Wang L. Qu S. Sheng X. Kristian A. Mælandsmo G.M. Pällmann N. Yuca E. Tekedereli I. Gorgulu K. Alpay N. Sood A. Lopez‐Berestein G. Fazli L. Rennie P. et al.STAMP2 increases oxidative stress and is critical for prostate cancer.EMBO Mol. Med. 2015; 7 (25680860): 315-33110.15252/emmm.201404181Crossref PubMed Scopus (37) Google Scholar). At the molecular level, the four STEAP proteins are predicted to adopt a common architecture with intracellular N and C termini, six transmembrane helices, and a single heme B prosthetic group bound in the transmembrane domain (TMD) (26Kleven M.D. Dlakić M. Lawrence C.M. 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.J. Biol. Chem. 2015; 290 (26205815): 22558-2256910.1074/jbc.M115.664565Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). STEAP2–STEAP4 also contain an intracellular oxidoreductase domain (OxRD) that binds NADPH (27Sendamarai 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.Proc. Natl. Acad. Sci. U.S.A. 2008; 105 (18495927): 7410-741510.1073/pnas.0801318105Crossref PubMed Scopus (65) Google Scholar, 28Gauss 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.J. Biol. Chem. 2013; 288 (23733181): 20668-2068210.1074/jbc.M113.479154Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The ferric and cupric reductase mechanism of STEAP2–STEAP4 is defined by electron transfer from intracellular NADPH through membrane-embedded FAD and heme cofactors to chelated metal-ion complexes at the membrane extracellular side (26Kleven M.D. Dlakić M. Lawrence C.M. 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.J. Biol. Chem. 2015; 290 (26205815): 22558-2256910.1074/jbc.M115.664565Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 29Oosterheert W. van Bezouwen L.S. Rodenburg R.N.P. Granneman J. Förster F. Mattevi A. Gros P. Cryo-EM structures of human STEAP4 reveal mechanism of iron(III) reduction.Nat. Commun. 2018; 9: 433710.1038/s41467-018-06817-7Crossref PubMed Scopus (24) Google Scholar). In contrast to STEAP2–STEAP4, STEAP1 does not exhibit metalloreductase activity when overexpressed on mammalian cells (19Ohgami R.S. Campagna D.R. McDonald A. Fleming M.D. The Steap proteins are metalloreductases.Blood. 2006; 108 (16609065): 1388-139410.1182/blood-2006-02-003681Crossref PubMed Scopus (433) Google Scholar), suggesting that it may have a distinct yet unidentified function. However, a recent study revealed that dithionite-reduced, purified STEAP1 retains heme and is capable of reducing metal-ion complexes and oxygen (30Kim K. Mitra S. Wu G. Berka V. Song J. Yu Y. Poget S. Wang D.-N. Tsai A.-L. Zhou M. Six-transmembrane epithelial antigen of prostate 1 (STEAP1) has a single b heme and is capable of reducing metal ion complexes and oxygen.Biochemistry. 2016; 55 (27792302): 6673-668410.1021/acs.biochem.6b00610Crossref PubMed Scopus (31) Google Scholar), indicating that the absence of a binding site for an electron-donating substrate like NADPH could explain the lack of reductase activity for STEAP1. It has been proposed that STEAP1 may have a functional role in heterooligomeric complexes with other STEAP paralogues (19Ohgami R.S. Campagna D.R. McDonald A. Fleming M.D. The Steap proteins are metalloreductases.Blood. 2006; 108 (16609065): 1388-139410.1182/blood-2006-02-003681Crossref PubMed Scopus (433) Google Scholar, 30Kim K. Mitra S. Wu G. Berka V. Song J. Yu Y. Poget S. Wang D.-N. Tsai A.-L. Zhou M. Six-transmembrane epithelial antigen of prostate 1 (STEAP1) has a single b heme and is capable of reducing metal ion complexes and oxygen.Biochemistry. 2016; 55 (27792302): 6673-668410.1021/acs.biochem.6b00610Crossref PubMed Scopus (31) Google Scholar). In support of this, its expression often correlates with the expression of STEAP2 in cancers (17Grunewald T.G.P. Bach H. Cossarizza A. Matsumoto I. The STEAP protein family: versatile oxidoreductases and targets for cancer immunotherapy with overlapping and distinct cellular functions.Biol. Cell. 2012; 104 (22804687): 641-65710.1111/boc.201200027Crossref PubMed Scopus (62) Google Scholar) and both proteins co-purify in detergent (30Kim K. Mitra S. Wu G. Berka V. Song J. Yu Y. Poget S. Wang D.-N. Tsai A.-L. Zhou M. Six-transmembrane epithelial antigen of prostate 1 (STEAP1) has a single b heme and is capable of reducing metal ion complexes and oxygen.Biochemistry. 2016; 55 (27792302): 6673-668410.1021/acs.biochem.6b00610Crossref PubMed Scopus (31) Google Scholar), suggesting that they could form a functional complex. Further indications for a functional heterotrimeric STEAP complex emerged from the recent cryo-EM structures of homotrimeric human STEAP4 (29Oosterheert W. van Bezouwen L.S. Rodenburg R.N.P. Granneman J. Förster F. Mattevi A. Gros P. Cryo-EM structures of human STEAP4 reveal mechanism of iron(III) reduction.Nat. Commun. 2018; 9: 433710.1038/s41467-018-06817-7Crossref PubMed Scopus (24) Google Scholar), 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. However, the in vivo redox activity of STEAP1, in both the absence and presence of other STEAP paralogues, remains to be established. In addition, there are no high-resolution structures available to help distinguish a functional role for STEAP1 as a metalloreductase or, as previously proposed, a potential channel or transporter protein (1Hubert 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.Proc. Natl. Acad. Sci. U.S.A. 1999; 96 (10588738): 14523-1452810.1073/pnas.96.25.14523Crossref PubMed Scopus (269) Google Scholar, 2Gomes I.M. Maia C.J. Santos C.R. STEAP proteins: from structure to applications in cancer therapy.Mol. Cancer Res. 2012; 10 (22522456): 573-58710.1158/1541-7786.MCR-11-0281Crossref PubMed Scopus (117) Google Scholar, 5Challita-Eid P.M. Morrison K. Etessami S. An Z. Morrison K.J. Perez-Villar J.J. Raitano A.B. Jia X.C. Gudas J.M. Kanner S.B. Jakobovits A. Monoclonal antibodies to six-transmembrane epithelial antigen of the prostate-1 inhibit intercellular communication in vitro and growth of human tumor xenografts in vivo.Cancer Res. 2007; 67 (17575147): 5798-580510.1158/0008-5472.CAN-06-3849Crossref PubMed Scopus (73) Google Scholar, 31Yamamoto T. Tamura Y. Kobayashi J. Kamiguchi K. Hirohashi Y. Miyazaki A. Torigoe T. Asanuma H. Hiratsuka H. Sato N. Six-transmembrane epithelial antigen of the prostate-1 plays a role for in vivo tumor growth via intercellular communication.Exp. Cell Res. 2013; 319 (23916873): 2617-262610.1016/j.yexcr.2013.07.025Crossref PubMed Scopus (22) Google Scholar). Thus, although STEAP1 is a populous plasma membrane component of many different types of cancer cells and hence is a promising novel therapeutic target, its structure and function in both health and disease remain unknown. Here, we present the cryo-EM structure of full-length, trimeric human STEAP1 bound to three Fab fragments of the therapeutically relevant mAb120.545. The Fabs dock on the extracellular helices of STEAP1 through an extensive polar interface. The TMD of STEAP1 resembles the architecture of the STEAP4 TMD and exhibits cellular ferric reductase activity when fused to the NADPH-binding OxRD of STEAP4. A previous pioneering study reported the biophysical and electrochemical characterization of N-terminally truncated rabbit STEAP1, purified from insect cells in lauryl maltose neopentyl glycol detergent (30Kim K. Mitra S. Wu G. Berka V. Song J. Yu Y. Poget S. Wang D.-N. Tsai A.-L. Zhou M. Six-transmembrane epithelial antigen of prostate 1 (STEAP1) has a single b heme and is capable of reducing metal ion complexes and oxygen.Biochemistry. 2016; 55 (27792302): 6673-668410.1021/acs.biochem.6b00610Crossref PubMed Scopus (31) Google Scholar). Our initial attempts to purify full-length, human STEAP1 from mammalian HEK cells using a similar protocol were hampered by the loss of the noncovalently bound heme B cofactor during the purification, suggesting that STEAP1 was not natively folded. Therefore, we screened several other detergents for the solubilization of STEAP1 and identified digitonin as a suitable replacement for lauryl maltose neopentyl glycol. In digitonin, the purified protein retained its heme cofactor (Fig. 1A) and eluted as a monodisperse peak in size-exclusion chromatography experiments (Fig. 1, B and D). In addition, thermostability assays revealed a melting temperature of ∼55.5 °C for STEAP1 in digitonin (Fig. 1, D and E), indicating that the protein was stable outside its native membrane environment. To assess whether the extracellular domains of purified STEAP1 adopted a conformation similar to those of membrane-embedded STEAP1, we tested the binding of STEAP1 to the Fab fragment of mAb120.545, which exhibits 1 nm affinity for STEAP1 on cells (5Challita-Eid P.M. Morrison K. Etessami S. An Z. Morrison K.J. Perez-Villar J.J. Raitano A.B. Jia X.C. Gudas J.M. Kanner S.B. Jakobovits A. Monoclonal antibodies to six-transmembrane epithelial antigen of the prostate-1 inhibit intercellular communication in vitro and growth of human tumor xenografts in vivo.Cancer Res. 2007; 67 (17575147): 5798-580510.1158/0008-5472.CAN-06-3849Crossref PubMed Scopus (73) Google Scholar) and recognizes a conformation-dependent, nonlinear epitope (32Genentech Inc. (February, 18, 2015) Antibodies and immunoconjugates and uses therefor. U.S. Patent EP2502938B1.Google Scholar). Size-exclusion chromatography assays revealed a smaller elution volume for STEAP1 when it was premixed with Fab120.545 (Fig. 1B), suggesting the formation of a complex, which was then confirmed by SDS-PAGE analysis of the eluted sample (Fig. 1C). Thus, the conformation of the STEAP1 epitope recognized by the Fab fragment on cells is preserved during the detergent solubilization and purification of STEAP1. To gain insights into the molecular architecture of STEAP1, we set out to obtain a structural model of the protein using single-particle cryo-EM. Full-length, trimeric STEAP1 proved to be a challenging sample for EM due to its small size (<120 kDa) and the absence of folded domains protruding from the membrane region. To create a larger particle with more extramembrane features to facilitate EM image processing, we opted to determine the structure of STEAP1 purified in complex with Fab120.545. The complementarity-determining regions of Fab120.545 are identical to those present in STEAP1 antibodies used in clinical trials (Fig. S1), indicating that the structure of the STEAP1-Fab120.545 complex could also be useful in engineering antibodies and other molecules that target STEAP1 in cancer. Micrographs collected on a 200-kV Talos Arctica microscope showed nonaggregated particles distributed in vitreous ice (Fig. S2A). Subsequent 2D classification experiments yielded class averages with clear secondary structure elements and furthermore revealed that more than one Fab fragment is bound to micelle-embedded STEAP1 (Fig. 2A; Fig. S2B). Image processing in RELION (33Scheres S.H.W. RELION: implementation of a Bayesian approach to cryo-EM structure determination.J. Struct. Biol. 2012; 180 (23000701): 519-53010.1016/j.jsb.2012.09.006Crossref PubMed Scopus (3034) Google Scholar) finally resulted in a reconstructed cryo-EM-density map at ∼3.0-Å resolution (Fig. 2B; Fig. S2, C–F). The map displayed well-defined side chain density for the TMD of STEAP1 and the variable regions of the Fab (Fig. S3). The model for STEAP1 was built with the TMD of STEAP4 as the template (29Oosterheert W. van Bezouwen L.S. Rodenburg R.N.P. Granneman J. Förster F. Mattevi A. Gros P. Cryo-EM structures of human STEAP4 reveal mechanism of iron(III) reduction.Nat. Commun. 2018; 9: 433710.1038/s41467-018-06817-7Crossref PubMed Scopus (24) Google Scholar) (PDB code 6HCY), whereas the starting model for the Fab was generated through the PIGS homology server (34Marcatili P. Rosi A. Tramontano A. PIGS : automatic prediction of antibody structures.Bioinformatics. 2008; 24: 1953-195410.1093/bioinformatics/btn341Crossref PubMed Scopus (146) Google Scholar). The refined structure has acceptable stereochemistry and exhibits high correlation to the cryo-EM density map within the determined resolution (Fig. S3, G and H; Table 1).Table 1Cryo-EM data collection, refinement, and validation statistics for STEAP1-Fab120.545 (PDB code 6Y9B, EMDB-10735)Data collection and processingMicroscopeTalos ArcticaCameraGatan K2 Summit + GIF filterMagnification×130,000Voltage (kV)200Frame/total exposure time (s)0.25/6.5No. of frames26Electron exposure (e−/Å2)49.5Defocus range (µm)−0.5 to −3.0Pixel size (Å)1.029Symmetry imposedC3No. of micrographs5,325No. of initial particle images616,302No. of final particles images169,426Map resolution (FSC = 0.143 threshold) (Å)2.97Map resolution range (Å)2.9–5.0RefinementModel resolution (FSC = 0.5 threshold) (Å)3.04Map sharpening B factor (Å2)0Model compositionNo. of nonhydrogen atoms11,889No. of protein residues1,434No. of ligands6B-factors (Å2)Protein100.5Ligands92.8RMSDBond lengths (Å)0.004Bond angles (°)0.536ValidationMolProbity score1.69Clash score5.08Poor rotamers (%)1.2Ramachandran plotFavored (%)94.6Allowed (%)5.4Disallowed (%)0 Open table in a new tab The cryo-EM structure reveals a 1:1 stoichiometry of the STEAP1-Fab120.545 complex, with 3 STEAP1 protomers interacting with 3 Fab molecules (Fig. 2, B–D). The Fabs bind at the extracellular region of STEAP1, consistent with the observation that the antibody targets STEAP1 expressed on intact cancer cells. The intracellular loops of STEAP1 extend ∼18 Å from the membrane region into the cytoplasm, whereas the Fabs protrude up to ∼75 Å into the extracellular space. STEAP1 adopts a trimeric arrangement that is similar to that of its family member STEAP4 (41% amino acid sequence identity; root mean square deviation (RMSD) of 0.8 Å for 640 Cα atoms) (Fig. 3). Each STEAP1 subunit contains six membrane-spanning α-helices (h1–h6) that define the TMD of the protein. A single B-type heme cofactor is surrounded by helices, h2, h3, h4, and h5 at the extracellular membrane leaflet (Fig. S3B). Strictly conserved histidine residues H175 and H268 coordinate the central iron moiety of the heme prosthetic group, thereby resembling the hexacoordinated heme arrangement of STEAP4. At the intracellular membrane leaflet side of the TMD, we observed weak density not corresponding to any protein residues. An overlay with the structure of STEAP4 revealed that the observed density overlaps with the flavin ring of the FAD-binding site in STEAP4 (Fig. S3D). The FAD-interacting residues in the TMD of STEAP3 and 4 are conserved in STEAP1, and the STEAP1-Fab120.545 cryo-EM sample was supplemented with 1 mm FAD before grid freezing. However, STEAP3 and STEAP4 also interact with the adenine moiety of FAD via their intracellular OxRD (26Kleven M.D. Dlakić M. Lawrence C.M. 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.J. Biol. Chem. 2015; 290 (26205815): 22558-2256910.1074/jbc.M115.664565Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 29Oosterheert W. van Bezouwen L.S. Rodenburg R.N.P. Granneman J. Förster F. Mattevi A. Gros P. Cryo-EM structures of human STEAP4 reveal mechanism of iron(III) reduction.Nat. Commun. 2018; 9: 433710.1038/s41467-018-06817-7Crossref PubMed Scopus (24) Google Scholar) (Fig. 3B; Fig. S3D), which is missing in STEAP1. In line with this, STEAP3 and STEAP4 exhibit a low micromolar affinity for FAD (Kd = ∼1 μm) (26Kleven M.D. Dlakić M. Lawrence C.M. 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.J. Biol. Chem. 2015; 290 (26205815): 22558-2256910.1074/jbc.M115.664565Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 29Oosterheert W. van Bezouwen L.S. Rodenburg R.N.P. Granneman J. F
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