MitoNEET is a uniquely folded 2Fe 2S outer mitochondrial membrane protein stabilized by pioglitazone

Abstract

Iron-sulfur (Fe-S) proteins are key players in vital processes involving energy homeostasis and metabolism from the simplest to most complex organisms. We report a 1.5 A x-ray crystal structure of the first identified outer mitochondrial membrane Fe-S protein, mitoNEET. Two protomers intertwine to form a unique dimeric structure that constitutes a new fold to not only the approximately 650 reported Fe-S protein structures but also to all known proteins. We name this motif the NEET fold. The protomers form a two-domain structure: a beta-cap domain and a cluster-binding domain that coordinates two acid-labile 2Fe-2S clusters. Binding of pioglitazone, an insulin-sensitizing thiazolidinedione used in the treatment of type 2 diabetes, stabilizes the protein against 2Fe-2S cluster release. The biophysical properties of mitoNEET suggest that it may participate in a redox-sensitive signaling and/or in Fe-S cluster transfer.

Fig. 1.

Overall structural organization and domain topology of dimeric mitoNEET. (A Upper) The backbone tracing of each protomer colored in green and magenta, respectively, together with the 2F_o − F_c electron density (gray) map contoured at 1.5σ. The protomers pack in a parallel fashion with each protomer harboring a 2Fe–2S cluster, depicted as yellow (sulfur) and red (iron) spheres; N and C termini are indicated. (Lower) The box shows an expanded view of one 2Fe–2S cluster (rotated ≈90° from upper view) and ligands and the corresponding 2F_o − F_c electron density (gray) map contoured at 2.0σ. (B) Ribbon diagram highlighting the two domains of the mitoNEET dimer. A six-stranded β-sandwich forms the intertwined β-cap domain and a larger cluster-binding domain carries two 2Fe–2S clusters. (C) A topology diagram highlighting the organization of the secondary structural units (numbered) illustrates the strand swap between protomers. (D) Coded segments contributing to each domain are highlighted on the primary sequence and block diagram. Protomer sequences within the cluster-binding domain are colored in purple and dark green, and the sequences corresponding to the β-cap domain are given in pink and light green, respectively. The amino acid sequence of the resolved amino acid strand is shown in the box with the cluster and cap regions colored as for protomer A; the numbers indicate the first (Lys-42) and last (Lys-106) resolved amino acid. The ligands to the 2Fe–2S cluster shown in the expanded boxed view in A are indicated in bold and highlighted in gray. The 2Fe–2S binding cradle is located sequentially between two partial β-cap domains. Rendered with Pymol (11).

Fig. 2.

The overall distribution of charges within mitoNEET creates a macrodipole separated by a hydrophobic belt. (A) The ribbon diagram of mitoNEET is displayed in two orientations. The Lower view is rotated 90° from the top along the vertical axis shown in the center. The protomers of the unit are colored in green and purple, respectively. Each 2Fe–2S cluster is shown with yellow (sulfur) and red (iron) spheres. (B) The ribbons of each protomer are colored gray, and the packing of the 10 aromatic residues (5 from each protomer) are emphasized by yellow dots. Apolar residues are also localized to this region but are not shown. (C) The separation of charged residues in mitoNEET indicates segregation of the aromatic and apolar regions of the protein. The negatively charged residues are labeled in red, and the positively charged residues are labeled in blue. The Upper panel emphasizes both the asymmetry of charges within the interior of the molecule and the separation of these charges by the nonpolar residues (B).

Fig. 3.

The 2Fe–2S cluster-binding cradle. (Upper) View of the 2Fe–2S cluster (Fe as brown and S as yellow spheres) from a perspective rotated ≈15° from that shown in the Upper panel of Fig. 2. The amino acids belonging to the individual protomers are shown in green and magenta. The two 2Fe–2S cradles are related to each other via a 180° rotation along the C₂ symmetry axis of the dimer. Cys-83 and His-87 bind the outermost Fe, whereas the innermost Fe is bound by Cys-72 and Cys-74. The solvent-accessible His-87 is located at the end of the prominent α-helix in the cluster-binding domain (Fig. 1). (Lower) View of the cluster cradle rotated 90° clockwise from the view presented in Upper. Two additional residues, Arg-73 and His-58, form an unusual His–Arg interprotomer hydrogen bond within the interior of the protein dimer. The distances between the nitrogen of His-58 and the guanidinium nitrogen atoms of Arg-73 are indicated. The two symmetry-related Arg form the positive end of the internal macrodipole (Fig. 2C).

Fig. 4.

The binding of pioglitazone to mitoNEET stabilizes the Fe–S cluster. (A) The stability of the 2Fe–2S cluster of mitoNEET is increased in the presence of pioglitazone. The change in the signature absorbance spectrum (460 nm) of the 2Fe–2S cluster (oxidized form) was monitored as a function of time at pH 6.0 in the absence and presence of stoichiometric pioglitazone (15 μM). The binding of the insulin-sensitizing drug pioglitazone increased the observed half-life by ≈10-fold. (B) 1D vectors derived from 2D homonuclear ¹H NOESY spectra of mitoNEET, with and without pioglitazone (D₂O, pH* 7.8, 35°C) are shown. The 1D vectors are along ω₁ at the ω₂ chemical shift typical of the aromatic ring protons of Trp and/or Phe residues.

Fig. 5.

Possible functional implications of mitoNEET's biophysical properties. MitoNEET is shown linked (magenta and green) to the OMM (gray) (not to scale). On the basis of mitoNEET biophysical properties, two possible functions are suggested: cluster transfer (blue arrows) and electron transfer (wine arrows). Right side (wine), our previous results (9) showed that the 2Fe–2S cluster could be reduced (1′) and reoxidized (2′) (−0.3 V ≤ Em ≤ 0.1 V). Left side, previous and current results showed that upon protonation of His-87 (1), the 2Fe–2S cluster dissociates from the protein (2). We here propose that the changes in the interaction of His-87 with the cluster are likely related to its function. In vivo this interaction may be broken by docking of another protein, thereby providing a convenient trigger for cluster release. Binding of pioglitazone to mitoNEET (Fig. 4) increases the stability of the 2Fe–2S cluster, thereby inhibiting release of the cluster.