Crystallographic Studies of Human MitoNEET

Abstract

MitoNEET was identified as an outer mitochondrial membrane protein that can potentially bind the anti-diabetes drug pioglitazone. The crystal structure of the cytoplasmic mitoNEET (residues 33–108) is determined in this study. The structure presents a novel protein fold and contains a [2Fe-2S] cluster-binding domain. The [2Fe-2S] cluster is coordinated to the protein by Cys-72, Cys-74, Cys-83, and His-87 residues. This coordination is also novel compared with the traditional [2Fe-2S] cluster coordinated by four cysteines or two cysteines and two histidines. The cytoplasmic mitoNEET forms homodimers in solution and in crystal. The dimerization is mainly mediated by hydrophobic interactions as well as hydrogen bonds coordinated by two water molecules binding at the interface. His-87 residue, which plays an important role in the coordination of the [2Fe-2S] cluster, is exposed to the solvent on the dimer surface. It is proposed that mitoNEET dimer may interact with other proteins via the surface residues in close proximity to the [2Fe-2S] cluster. MitoNEET was identified as an outer mitochondrial membrane protein that can potentially bind the anti-diabetes drug pioglitazone. The crystal structure of the cytoplasmic mitoNEET (residues 33–108) is determined in this study. The structure presents a novel protein fold and contains a [2Fe-2S] cluster-binding domain. The [2Fe-2S] cluster is coordinated to the protein by Cys-72, Cys-74, Cys-83, and His-87 residues. This coordination is also novel compared with the traditional [2Fe-2S] cluster coordinated by four cysteines or two cysteines and two histidines. The cytoplasmic mitoNEET forms homodimers in solution and in crystal. The dimerization is mainly mediated by hydrophobic interactions as well as hydrogen bonds coordinated by two water molecules binding at the interface. His-87 residue, which plays an important role in the coordination of the [2Fe-2S] cluster, is exposed to the solvent on the dimer surface. It is proposed that mitoNEET dimer may interact with other proteins via the surface residues in close proximity to the [2Fe-2S] cluster. MitoNEET is a protein recently identified to be localized on the outer membrane of mitochondria (1Wiley S.E. Murphy A.N. Ross S.A. van der Geer P. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 5318-5323Crossref PubMed Scopus (218) Google Scholar). It was first identified as a protein cross-linked with photoaffinity probe of pioglitazone, thus appearing to be a pharmacological target of pioglitazone. It was named based on its localization in mitochondria and the Asn-Glu-Glu-Thr (NEET) sequence in its C-terminal domain (2Colca J.R. McDonald W.G. Waldon D.J. Leone J.W. Lull J.M. Bannow C.A. Lund E.T. Mathews W.R. Am. J. Physiol. 2004; 286: E252-E260Crossref PubMed Scopus (270) Google Scholar). Pioglitazone is a member of the class of thiazolidinediones (TZDs) 3The abbreviations used are: TZD, thiazolidinedione; PDB, Protein Data Bank; SAD, single anomalous dispersion; MES, 4-morpholineethanesulfonic acid. 3The abbreviations used are: TZD, thiazolidinedione; PDB, Protein Data Bank; SAD, single anomalous dispersion; MES, 4-morpholineethanesulfonic acid. that are insulin sensitizers for treatment of type II diabetes (3Aronoff S. Rosenblatt S. Braithwaite S. Egan J.W. Mathisen A.L. Schneider R.L. Diabetes Care. 2000; 23: 1605-1611Crossref PubMed Scopus (585) Google Scholar). It has been shown that pioglitazone functions as a ligand of peroxisome proliferator-activated receptor γ (4Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. Willson T.M. Kliewer S.A. J. Biol. Chem. 1995; 270: 12953-12956Abstract Full Text Full Text PDF PubMed Scopus (3456) Google Scholar). However, additional peroxisome proliferator-activated receptor γ-independent mechanisms have been suggested for the clinical effects of pioglitazone, including its actions on the mitochondrial function (5Feinstein D.L. Spagnolo A. Akar C. Weinberg G. Murphy P. Gavrilyuk V. Dello Russo C. Biochem. Pharmacol. 2005; 70: 177-188Crossref PubMed Scopus (239) Google Scholar). Wiley et al. (1Wiley S.E. Murphy A.N. Ross S.A. van der Geer P. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 5318-5323Crossref PubMed Scopus (218) Google Scholar) recently reported that mitoNEET could negatively regulate electron transport and oxidative capacity of mitochondria since an apparent decrease of the complex I-dependent oxygen consumption was observed in the mitochondria isolated from the mitoNEET-deficient mice heart. Interestingly, pioglitazone was recently reported to have negative regulatory effect on complex I activity in muscle, liver, and astroglioma cells (6Brunmair B. Staniek K. Gras F. Scharf N. Althaym A. Clara R. Roden M. Gnaiger E. Nohl H. Waldhausl W. Furnsinn C. Diabetes. 2004; 53: 1052-1059Crossref PubMed Scopus (439) Google Scholar, 7Perez-Ortiz J.M. Tranque P. Burgos M. Vaquero C.F. Llopis J. Mol. Pharmacol. 2007; 72: 407-417Crossref PubMed Scopus (38) Google Scholar) and positive regulatory effect in neuron-like cells (8Ghosh S. Patel N. Rahn D. McAllister J. Sadeghi S. Horwitz G. Berry D. Wang K.X. Swerdlow R.H. Mol. Pharmacol. 2007; 71: 1695-1702Crossref PubMed Scopus (93) Google Scholar). This correlation and the mitochondrial localization of mitoNEET suggest that pioglitazone may exert its action on mitochondria by regulating the activities of respiratory complexes via its interaction with mitoNEET. Type II diabetes is a complex metabolic disease characterized by insulin resistance in the initial stage. The disease and the associated complications have become prevailing public health concerns; thus it is significant to determine the mechanisms by which the anti-diabetes drug pioglitazone functions as an insulin sensitizer. Moreover, it was recently reported that rosiglitazone, another Type II diabetes drug in the thiazolidinedione family that has similar binding affinity to mitoNEET, may increase the risk of myocardial infarction (9Home P.D. Pocock S.J. Beck-Nielsen H. Gomis R. Hanefeld M. Jones N.P. Komajda M. McMurray J.J. N. Engl. J. Med. 2007; 357: 28-38Crossref PubMed Scopus (723) Google Scholar, 10Nissen S.E. Wolski K. N. Engl. J. Med. 2007; 356: 2457-2471Crossref PubMed Scopus (4092) Google Scholar). In addition, pioglitazone has also been demonstrated to be a potential treatment of neurodegenerative diseases including multiple sclerosis (11Feinstein D.L. Galea E. Gavrilyuk V. Brosnan C.F. Whitacre C.C. Dumitrescu-Ozimek L. Landreth G.E. Pershadsingh H.A. Weinberg G. Heneka M.T. Ann. Neurol. 2002; 51: 694-702Crossref PubMed Scopus (279) Google Scholar, 12Pershadsingh H.A. Heneka M.T. Saini R. Amin N.M. Broeske D.J. Feinstein D.L. J. Neuroinflammation. 2004; 1: 3Crossref PubMed Scopus (72) Google Scholar, 13Klotz L. Schmidt M. Giese T. Sastre M. Knolle P. Klockgether T. Heneka M.T. J. Immunol. 2005; 175: 4948-4955Crossref PubMed Scopus (101) Google Scholar), Alzheimer disease (14Heneka M.T. Sastre M. Dumitrescu-Ozimek L. Hanke A. Dewachter I. Kuiperi C. O'Banion K. Klockgether T. Van Leuven F. Landreth G.E. Brain. 2005; 128: 1442-1453Crossref PubMed Scopus (510) Google Scholar), and amyotrophic lateral sclerosis (15Schutz B. Reimann J. Dumitrescu-Ozimek L. Kappes-Horn K. Landreth G.E. Schurmann B. Zimmer A. Heneka M.T. J. Neurosci. 2005; 25: 7805-7812Crossref PubMed Scopus (189) Google Scholar, 16Kiaei M. Kipiani K. Chen J. Calingasan N.Y. Beal M.F. Exp. Neurol. 2005; 191: 331-336Crossref PubMed Scopus (189) Google Scholar). Therefore, the structural studies of mitoNEET, which is a potential target of pioglitazone and the TZDs, are highly significant for diabetes and other human diseases. Here we report the crystal structure at 1.8 Å resolution of the cytoplasmic portion of human mitoNEET (from residues 33–108, referred as mitoNEET33–108). The structure of mitoNEET33–108 represents a new overall fold and contains a novel Cys3–His1 coordinated [2Fe-2S] cluster. The structure reveals a homodimer of mitoNEET33–108 with strong hydrophobic interactions and hydrogen bond network at the dimeric interface. The data suggest that it is highly likely that mitoNEET forms a dimer on the outer membrane of mitochondria. Protein Expression and Purification—The gene of human mitoNEE33–108 was amplified by PCR and cloned to the pMCSG9 vector using the ligation-independent cloning technology (17Donnelly M.I. Zhou M. Millard C.S. Clancy S. Stols L. Eschenfeldt W.H. Collart F.R. Joachimiak A. Protein Expression Purif. 2006; 47: 446-454Crossref PubMed Scopus (148) Google Scholar). His-maltose binding protein-tagged protein was produced in the Escherichia coli Rosetta (DE3) strains. Cells were grown at 37 °C in 3.2 liters of LB medium containing 100 μg/ml ampicillin. When A600 reached 0.6, the cells were induced at 16 °C for 20 h by adding 0.35 mm isopropyl-β-d-thiogalactoside. Then the cells were harvested by centrifugation at 4000 rpm for 30 min. The pellets were resuspended and sonicated in 60 ml of lysis buffer (20 mm Tris-HCl, pH 8.0, 500 mm NaCl). After centrifugation at 16,000 rpm for 30 min, the supernatant was loaded onto a Ni2+-affinity column equilibrated with the lysis buffer. The column was washed sequentially by 20 column volumes of binding buffer (20 mm Tris-HCl, pH 8.0, 20 mm imidazole), and 10 column volumes of washing buffer (20 mm Tris-HCl, pH 8.0, 40 mm imidazole), and then the protein was eluted with elution buffer (200 mm Tris-HCl, pH 8.0, 400 mm imidazole). The eluted solution was cleaved overnight with tobacco etch virus protease at 4 °C, which yields untagged mitoNEET33–108 with three residues (Ser-Asn-Ala) at the N terminus. The untagged mitoNEET33–108 was further purified by ion-exchange chromatography on a 5-ml HiTrap SP FF ion-exchange column. Fractions containing mitoNEET33–108 were desalted and concentrated to ∼10 mg/ml in the concentration buffer (20 mm Tris-HCl, pH 8.0) and stored at –20 °C. When being tentatively purified for the first time, the eluted solution after tobacco etch virus protease cleavage was subjected to the size exclusion chromatography before the ion-exchange chromatography, and the retention time suggested that mitoNEET33–108 protein existed in solution as a dimer. Crystallization and Data Collection—Crystals were obtained at 289 K using the hanging-drop vapor diffusion method. Each hanging drop is a 1:1 mixture of protein and reservoir solution. The crystal used for the single anomalous dispersion (SAD) experiment was grown in the reservoir solution containing 2.0 m NaCl and 0.1 m MES, pH 5.5, and the crystal used for the high resolution measurement was grown in the solution containing 2.0 m NaCl and 0.1 m Tris-HCl, pH 9.0. The SAD data set with high redundancy was collected at 100 K on a Rigaku R-AXIS IV++ imaging-plate system with a Rigaku FRE copper rotating-anode generator in Institute of Biophysics, Chinese Academy of Sciences. The data were processed with HKL2000 (18Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38526) Google Scholar). The high resolution data set was collected at beamline 19ID of the Structural Biology Center at the Advanced Photon Source in the Argonne National Laboratory with the ADSC315 CCD detector and were processed with HKL3000 (19Minor W. Cymborowski M. Otwinowski Z. Chruszcz M. Acta Crystallogr. Sect. D Biol. Crystallogr. 2006; 62: 859-866Crossref PubMed Scopus (1553) Google Scholar). The processing statistics are summarized in Table 1.TABLE 1Data collection, phasing, refinement statisticsSADHigh resolutionData collection Wavelength (Å)1.54180.97937 Space group1412214122 Unit cell dimensions (Å) and angles (°)a = b = 58.959, c = 175.231 α = β = γ = 90a = b = 58.794, c = 175.306, α = β = γ = 90 Resolution range (Å)50-2.3 (2.38-2.30)30-1.8 (1.86-1.80) No. of total reflections320731336253 No. of unique reflections727814738 Completeness (%)99.9 (100)99.4 (99.0) Redundancy14.6 (14.8)9.1 (8.3) I/σ(I)36.21 (9.00)33.80 (4.78) Rmerge (%)aRmerg = Σ|Ii — Im|/ΣIi, where Ii is the intensity of the measured reflection and Im is the mean intensity of all symmetry-related reflections.0.123 (0.466)0.073 (0.440) Wilson B-factor (Å2)bWilson B-factor is estimated by Truncate program in CCP4 (36).33.57.2SAD phasing Sites2 Resolution (Å)30-2.3 Phasing power (anomalous)cPhasing power = |FH(calc)|/|E|, where FH(calc) is the calculated heavy atom contribution and E is the lack of closure.0.926Structure refinement Resolution (Å)30-1.8 Rcryst/Rfree (%)dRcryst = Σ||Fobs| — |Fcalc||/Σ|Fobs|, where Fobs and Fcalc are observed and calculated structure factors. Rfree = ΣT||Fobs| — |Fcalc||/ΣT|Fobs|, where T is a test data set of about 10% of the total reflections randomly chosen and set aside prior to refinement.15.9/18.8 No. of reflections Work set13189 Test set699 r.m.s.d.er.m.s.d, root mean square deviation. from ideal values Bond length (Å)0.012 Bond angels (°)1.491 Average B-factor (Å2)27.30 No. of atoms Protein627 [2Fe-2S]4 Chloride ion1 Waters125Ramachandran plot Most favored regions (%)85.1 Additionally allowed (%)14.9a Rmerg = Σ|Ii — Im|/ΣIi, where Ii is the intensity of the measured reflection and Im is the mean intensity of all symmetry-related reflections.b Wilson B-factor is estimated by Truncate program in CCP4 (36Collaborative Computational Project, Number 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19748) Google Scholar).c Phasing power = |FH(calc)|/|E|, where FH(calc) is the calculated heavy atom contribution and E is the lack of closure.d Rcryst = Σ||Fobs| — |Fcalc||/Σ|Fobs|, where Fobs and Fcalc are observed and calculated structure factors. Rfree = ΣT||Fobs| — |Fcalc||/ΣT|Fobs|, where T is a test data set of about 10% of the total reflections randomly chosen and set aside prior to refinement.e r.m.s.d, root mean square deviation. Open table in a new tab Structure Determination—The crystal structure of human mitoNEET33–108 was solved by the SAD method using the anomalous signal of iron atoms. Heavy atom positions were located by SHLXD (20Schneider T.R. Sheldrick G.M. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 1772-1779Crossref PubMed Scopus (1576) Google Scholar). Heavy atom refinement, phasing, and density flattening were performed by autoSHARP (21Vonrhein C. Blanc E. Roversi P. Bricogne G. Methods Mol. Biol. 2006; 364: 215-230Google Scholar). The initial model was automatically built by ARP/wARP (22Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2563) Google Scholar), manually modified with Coot (23Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23226) Google Scholar), and refined with Refmac5 (24Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13854) Google Scholar). The [2Fe-2S] cluster and water molecules were built in according to the Fo – Fc map at the very late stage of refinement. The stereochemical quality of the final model was evaluated by Procheck (25Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Structure determination and refinement statistics are in Table final model refined to resolution of of mitoNEET and at the N from the [2Fe-2S] cluster, and water was as and three C-terminal residues and were to be to the electron The [2Fe-2S] cluster and are identified based on the anomalous map using SAD The – Fc and map the [2Fe-2S] cluster and the are shown in The model is well and all the data statistics are shown in Table The structure contains a from to in the and a for the of the protein and [2Fe-2S] cluster is in the and and is by the and the The is from the cluster-binding domain (residues from to and with the cluster-binding domain mainly by hydrophobic residues as and structural in the with the program two with the of and that the mitoNEET33–108 structure represents a new The structure are shown in In addition, we two human proteins and using protein sequence on The localization of as was reported (1Wiley S.E. Murphy A.N. Ross S.A. van der Geer P. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 5318-5323Crossref PubMed Scopus (218) Google Scholar), the structure and function of two proteins are to on the sequence we that two proteins are likely to fold as mitoNEET, and the sequence is shown in In is a new that has been reported has the length as mitoNEET and sequence with mitoNEET. It to be determined the new is a unique protein in human structure and function also to be [2Fe-2S] [2Fe-2S] cluster is coordinated by Cys-72, Cys-74, and from the and and by His-87 from the of and [2Fe-2S] in the can be two coordinated and with two cysteines and two The structure of a [2Fe-2S] cluster coordinated with Cys3–His1 as in this of mitoNEET is the first time that this novel structure of [2Fe-2S] cluster is demonstrated in crystal the coordination the [2Fe-2S] cluster is to the [2Fe-2S] the two and the are in a and the are coordinated The cluster-binding domain of mitoNEET was proposed as a domain based on the sequence the structural determination in this we proposed to this domain as the class of [2Fe-2S] cluster-binding domain. In with the for coordinated [2Fe-2S] in R. R. A. D.J. E. U. 2006; PubMed Scopus Google Scholar) and for coordinated [2Fe-2S] cluster J.R. M. J.M. Appl. 1999; PubMed Google Scholar), the coordinated residues in mitoNEET an of which in the unique of the [2Fe-2S] cluster-binding domain. coordinated residues are close to another that the and the [2Fe-2S] cluster in the cluster-binding domain In the of the the coordinated residues and His-87 are exposed to the where two water molecules interact with the oxygen of and the of His-87 by hydrogen The crystal structure of the [2Fe-2S] cluster is also by a mitoNEET that a [2Fe-2S] cluster from the traditional in and protein S.E. L. van der Geer P. R. Murphy A.N. Dixon J.E. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, T. R. G.E. W.R. T.A. E. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The also that the [2Fe-2S] cluster coordination to mitoNEET is S.E. L. van der Geer P. R. Murphy A.N. Dixon J.E. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). crystal structure of mitoNEET33–108 dimer that the of His-87 is exposed to solvent and is to the from the other the on the dimeric of the pH of the of the in and the close proximity of His-87 to a residue, structural for the reported The Structure of crystal structure also reveals a homodimer of which is with the retention time on the size exclusion chromatography protein is in an homodimer is by the c of the hydrophobic residues in mitoNEET33–108 in the dimer and the dimerization of the total surface in The forms a β with from the other two are in the dimer as and from from the and from from the and as well as and from a of hydrophobic interactions the Therefore, the dimerization be very and could be for mitoNEET that mitoNEET as a dimer the Interestingly, the dimer is mainly two water molecules are in the hydrophobic of the dimer interface. water molecules the two by hydrogen bonds with the of and the oxygen atoms of and from the other water molecules can the of interactions Struct. 2006; PubMed Google Scholar). In the structure of mitoNEET33–108 the two water molecules the well to the hydrophobic interface. also the two by hydrogen In addition, it is reported that to J. 2005; PubMed Scopus Google Scholar). In the structure of mitoNEET33–108 the of the two is which is a for potential electron the two the electron has been The structural is with the proposed role of mitoNEET in regulating electron in mitochondria (1Wiley S.E. Murphy A.N. Ross S.A. van der Geer P. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 5318-5323Crossref PubMed Scopus (218) Google Scholar). the role of in the dimeric we that this is to the structure and function of mitoNEET. are this by to other residues. also solved a mitoNEET33–108 structure with a crystal in another Å data in which the dimer was the dimer was by the in This structure additional for the observed mitoNEET dimer as the dimer. The residues are reported to mitoNEET to the outer mitochondrial membrane (1Wiley S.E. Murphy A.N. Ross S.A. van der Geer P. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 5318-5323Crossref PubMed Scopus (218) Google Scholar). R. V. PubMed Scopus Google Scholar), residues from to are to a domain. In mitoNEET33–108 residues from to are from the In the the N of the two to the It is to that this dimer on the mitochondrial surface with the two the outer membrane in The surface potential of the dimer is shown in the His-87 that is to the [2Fe-2S] cluster binding in a on the protein surface. The on of the dimer may a for with other proteins and molecules as pioglitazone. the is in close proximity to the [2Fe-2S] cluster, the binding and potential of the [2Fe-2S] cluster be by the proteins and molecules that can potentially interact with the This the structure of mitoNEET and its potential function on the mitochondrial outer on the structure of mitoNEET33–108 in this it is likely that the binding of pioglitazone or the cluster binding and dimeric structure of mitoNEET as well as its function in mitochondria. The structural of the interaction mitoNEET and pioglitazone as well as other is and in the Institute of for the SAD data collection and the high resolution data collection, The high resolution data were collected at beamline 19ID of the Structural Biology Center at the Advanced Photon Source in the Argonne National The of in the data collection and data processing is highly The Argonne National Laboratory is by the of for the of of and