Crystallographic Studies of Human MitoNEET*

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.

lidinediones (TZDs) 3 that are insulin sensitizers for treatment of type II diabetes (3). It has been shown that pioglitazone functions as a ligand of peroxisome proliferator-activated receptor ␥ (4). However, additional peroxisome proliferator-activated receptor ␥-independent mechanisms have been suggested for the clinical effects of pioglitazone, including its actions on the mitochondrial function (5). Wiley et al. (1) 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 (6,7) and positive regulatory effect in neuron-like cells (8). 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 (9,10). In addition, pioglitazone has also been demonstrated to be a potential treatment of neurodegenerative diseases including multiple sclerosis (11)(12)(13), Alzheimer disease (14), and amyotrophic lateral sclerosis (15,16). 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 mitoNEET  ). The structure of mitoNEET 33-108 represents a new overall fold and contains a novel Cys 3 -His 1 coordinated [2Fe-2S] cluster. The structure reveals a homodimer of mitoNEET 33-108 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.

MATERIALS AND METHODS
Protein Expression and Purification-The gene of human mitoNEET  was amplified by PCR and cloned to the pMCSG9 vector using the ligation-independent cloning technology (17). 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 g/ml ampicillin. When A 600 reached 0.6, the cells were induced at 16°C for 20 h by adding 0.35 mM isopropyl-␤-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 Ni 2ϩ -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 mitoNEET 33-108 with three residues (Ser-Asn-Ala) at the N terminus. The untagged mitoNEET 33-108 was further purified by ion-exchange chromatography on a 5-ml HiTrap SP FF ion-exchange column. Fractions containing mitoNEET  were desalted and concentrated to ϳ10 mg/ml in the concentration buffer (20 mM Tris-HCl, pH 8.0) and stored at Ϫ20°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 mitoNEET 33-108 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 IVϩϩ 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 (18). 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 (19). The processing statistics are summarized in Table 1.
Structure Determination-The crystal structure of human mitoNEET 33-108 was solved by the SAD method using the anomalous signal of iron atoms. Heavy atom positions were located by SHLXD (20). Heavy atom refinement, phasing, and density flattening were performed by autoSHARP (21). The initial model was automatically built by ARP/wARP (22), manually modified with Coot (23), and refined with Refmac5 (24). The [2Fe-2S] cluster and water molecules were built in according to the F o Ϫ F c map at the very late stage of refinement. The stereochemical quality of the final model was evaluated by Procheck (25). Structure determination and refinement statistics are listed in Table 1.

RESULTS AND DISCUSSION
Overall Structure-The final model refined to 1.8Å resolution consists of 76 amino acids (Arg-33-Lys-105 of mitoNEET and Ser-Asn-Ala at the N terminus resulting from the vector), one [2Fe-2S] cluster, one chloride ion, and 125 water molecules.
Lys-105 was modeled as alanine, and three C-terminal residues (Lys-106, Glu-107, and Thr-108) were not able to be modeled due to the weak electron density. The [2Fe-2S] cluster and chloride ion are identified based on the anomalous difference Fourier map using SAD phases. The 2F o Ϫ F c and difference Fourier map around the [2Fe-2S] cluster and the chloride ion are shown in Fig. 1A. The model is well refined, and all the crystallographic data statistics are shown in Table 1.
The structure contains a long loop from Arg-33 to Lys-55 in the N-terminal part and a ␤-␤-␣-␤ topology for the rest of the protein (Fig. 1, B and C). A [2Fe-2S] cluster is bound in the loop between ␤2 and ␣1 and is flanked by the helix ␣1 and the two-stranded anti-parallel ␤-sheet (␤2␤3). The ␤1 strand is away from the cluster-binding domain (residues from ␤2 to ␤3) and interacts with the cluster-binding domain mainly by hydrophobic residues such as Ile-56, Phe-60, Ala-69, Tyr-71, Phe-80, and Phe-82. A structural homology search in the PDB with the program Dali gives two results with the Z-score of 0.5 and 0.4, respectively, indicating that the mitoNEET 33-108 structure represents a new fold. The secondary structure features are shown in Fig. 1C.
In addition, we found two human CDGSH domain-containing proteins (accession numbers A6NMV4 and Q8N5K1) using protein sequence similarity search (blastp on ExPASy against UniProt Knowledgebase). The subcellular localization of Q8N5K1 (named as "Miner 1") was reported (1), but the structure and function of these two proteins are unknown to date. Based on the sequence similarity, we predict that these two proteins are likely to fold similarly as mitoNEET, and the sequence alignment is shown in Fig.  1C. In particular, A6NMV4 is a new homolog that has not been reported previously. A6NMV4 has the identical length as mitoNEET and 87% sequence identity with mitoNEET. It remains to be determined whether the new homolog A6NMV4 is a unique protein expressed in human tissues. Its structure and function also need to be elucidated.
[2Fe-2S] Cluster-binding Domain-The [2Fe-2S] cluster is coordinated by Cys-72, Cys-74, and Cys-83 from the loop between ␤2 and ␣1 and by His-87 from the start point of ␣1 (Fig. 1, B and C). [2Fe-2S] clusters deposited in the PDB heretofore can be classified into two types: four-cysteine coordinated clusters and Rieske clusters with two cysteines and two histidines (Fig. 1D). The structure of a [2Fe-2S] cluster coordinated with Cys 3 -His 1 as found in this study of mitoNEET is the first time that this novel structure of [2Fe-2S] cluster is demonstrated in crystal structure. Except the coordination residues, the [2Fe-2S] cluster itself is identical to the classical [2Fe-2S] clusters; the two irons and the inorganic sulfurs are in a plane, and the irons are tetrahedrally coordinated (Fig. 1D).
The cluster-binding domain of mitoNEET was proposed as a CDGSH-type zinc finger domain based on the sequence information. With the three-dimensional structural determination in this study, we proposed to rename this domain as the third class of [2Fe-2S] cluster-binding domain. In comparison with the consensus motif Cys-X 4 -Cys-X 2 -Cys-X Ϸ30 -Cys for Cys 4 coordinated [2Fe-2S] clusters in ferredoxins (26) and Cys-X-His-X 15-17 -Cys-X 2 -His for Cys 2 -His 2 coordinated Rieske [2Fe-2S] cluster (27), the coordinated residues in mitoNEET present an arrangement of Cys-X-Cys-X 8 -Cys-X 3 -His, which results in the unique feature of the [2Fe-2S] cluster-binding domain.
These coordinated residues are so close to one another that the loop between ␤2 and ␣1 rolls up around the [2Fe-2S] cluster in the cluster-binding domain (Fig. 1B). In the open side of the loop, the coordinated residues Cys-83 and His-87 are partially exposed to the polar surroundings, where two water molecules interact respectively with the mainchain oxygen of Cys-83 and the ⑀2 nitrogen of His-87 by hydrogen bonds.
The crystal structure of the [2Fe-2S] cluster is also supported by a recent mitoNEET spectroscopic study that presented spectroscopic evidence suggesting a [2Fe-2S] cluster slightly different from the traditional ones found in ferredoxins and Rieske protein (28,29). The same study also showed that the [2Fe-2S] cluster coordination to mitoNEET is pH-dependent (28). Our crystal structure of mitoNEET 33-108 dimer shows that the side chain of His-87 is exposed to solvent and is adjacent to the basic residue Lys-55 from the other monomer (see the discussion on the dimeric structure). Because of the pH dependence of the protonation status of the imidazole ring in histidine and the close proximity of His-87 to a basic residue, our study provides detailed structural support for the reported pHdependent spectroscopic features.
The Dimeric Structure of mitoNEET-The crystal structure also reveals a homodimer of mitoNEET  , which is consistent with the retention time on the size exclusion chromatography during protein purification. There is one molecule in an asymmetric unit. A homodimer is formed by the crystallographic 2-fold axis c ( Fig. 2A). Approximately 58% of the hydrophobic residues in mitoNEET 33-108 exist in the dimer interface, and the dimerization buries 30% of the total surface area in each monomer. The N-terminal ␤1 strand forms a parallel ␤ sheet with ␤3 coming from the other monomer. Consequently, two symmetric three-stranded ␤-sheets are formed face-to-face in the dimer ( Fig. 2A). Residues such as Val-56 and Val-57 from ␤1, Leu-65 from the loop between ␤1 and ␤2, Tyr-71 from ␤2, Trp-75 from the loop between ␤2 and ␣1, as well as Leu-101 and Ile-103 from ␤3 provide a number of hydrophobic interactions between the monomers (Fig. 2B). Therefore, the dimerization should be very tight and could be necessary for mitoNEET functions. We propose that mitoNEET exists as a dimer under the physiological conditions. The anomalous difference Fourier density map (magenta) is contoured at 4 . The [2Fe-2S] cluster and the chloride ion are shown as spheres (iron is red; sulfur is yellow; Chlorine is green). The coordinated residues are shown as sticks (carbon is cyan; nitrogen is blue; sulfur is yellow; oxygen is orange). The dashed magenta line indicates the interaction between the chloride ion and the side chain of Asn-91. All images except panel C were prepared using the PyMOL program. B, ribbon diagram of the overall structure in stereo. The protein is colored by secondary structure elements (␣ helices are violet, ␤ strands are forest green, and a single turn of 3 10 helix is presented as a loop) and numbered from the N terminus to the C terminus. The [2Fe-2S] cluster and the side chains of the coordinated residues are shown as sticks and color-coded as in panel A. The three residues (Ser-Asn-Ala) from the expression vector at the N terminus are omitted in all the figures. C, the secondary structure of mitoNEET and sequence alignment with two human homologues. The secondary structure features of dimeric mitNEET 33-108 are shown as defined by DSSP (33). MitoNEET and two human CDGSH domain-containing proteins (A6NMV4 and Q8N5K1) are aligned using clustW (34). The strictly conserved residues are shown in red boxes; similar residues are shown in yellow boxes. Red triangles below the alignment indicate the cluster-coordinated residues, and the purple circles indicate the hydrophobic residues involved in dimerization. The residues of mitoNEET are indicated every 10 residues from Met-1 by black dots above the alignment. C was prepared with ESPript (35). Interestingly, although the dimer interface is mainly hydrophobic, two water molecules are symmetrically buried in the hydrophobic core of the dimer interface. These water molecules link the two monomers by hydrogen bonds with the side chain of Arg-73 and the main-chain oxygen atoms of Pro-81 and cluster-coordinated Cys-72 from the other monomer (Fig. 2C). Interfacial water molecules can enhance the stability of protein-protein interactions (30). In the structure of mitoNEET 33-108 dimer, the two interfacial water molecules make the basic Arg-73 well accommodated to the hydrophobic interface. They also stabilize the two monomers by intermonomer hydrogen bonds. In addition, it is reported that interfacial waters adjacent to redox centers influence their intermolecular electron-transfer reaction (31). In the structure of mitoNEET 33-108 dimer, the distance of the two clusters is ϳ14 Å, which is a reasonable distance for potential electron transfer between the two clusters, although the intersubunit electron transfer has not been experimentally tested yet. The structural feature, however, is consistent with the proposed role of mitoNEET in regulating electron transfer reactions in mitochondria (1). Given the special role of Arg-73 in the dimeric interface, we propose that this residue is critical to the structure and function of mitoNEET.
We are currently testing this hypothesis by substituting Arg-73 to other residues.
We also solved a mitoNEET 33-108 structure with a crystal in another space group (p2 1 2 1 2 1 , 3.0 Å resolution, data not shown), in which the same dimer was observed, although the dimer was formed by the non-crystallographic 2-fold symmetry in one asymmetric unit. This structure provides additional support for the observed mitoNEET dimer as representing the physiological dimer.
The N-terminal 32 residues are reported to direct mitoNEET to the outer mitochondrial membrane (1). Using GlobPlot (32),  residues from Trp-13 to Tyr-35 are predicted to constitute a transmembrane domain. In our mitoNEET 33-108 structure, residues from Ser-33 to Ala-43 are away from the dimerized core. In the dimer, the N terminus of the two monomers extends to the same direction ( Fig. 2A). It is reasonable to propose that this dimer would exist on the mitochondrial surface with the two N-terminal transmembrane domains inserted into the outer membrane in vivo.
The electrostatic surface potential of the dimer is shown in Fig. 2D. As discussed earlier, the His-87 residue that is critical to the [2Fe-2S] cluster binding resides in a basic patch on the protein surface. The basic patches on both sides of the dimer may provide a site for interacting with other proteins and small molecules such as pioglitazone. Since the basic patch is in close proximity to the [2Fe-2S] cluster, the binding properties and redox potential of the [2Fe-2S] cluster would be influenced by the proteins and small molecules that can potentially interact with the basic patches.
This study provides critical insights into the structure of mitoNEET and its potential function on the mitochondrial outer membrane. Based on the structure of mitoNEET  in this study, it is likely that the binding of pioglitazone or rosiglitazone would significantly change the Fe-S cluster binding and dimeric structure of mitoNEET as well as its function in mitochondria. The structural basis of the interaction between mitoNEET and pioglitazone as well as other TZD compounds is currently under investigation. Addendum-After the submission of this manuscript, two groups published the crystal structure of the cytoplasmic portion of mitoNEET independently (37,38).