Crystal structure of human mitoNEET reveals distinct groups of iron–sulfur proteins

Abstract

MitoNEET is a protein of unknown function present in the mitochondrial membrane that was recently shown to bind specifically the antidiabetic drug pioglizatone. Here, we report the crystal structure of the soluble domain (residues 32-108) of human mitoNEET at 1.8-Å resolution. The structure reveals an intertwined homodimer, and each subunit was observed to bind a [2Fe-2S] cluster. The [2Fe-2S] ligation pattern of three cysteines and one histidine differs from the known pattern of four cysteines in most cases or two cysteines and two histidines as observed in Rieske proteins. The [2Fe-2S] cluster is packed in a modular structure formed by 17 consecutive residues. The cluster-binding motif is conserved in at least seven distinct groups of proteins from bacteria, archaea, and eukaryotes, which show a consensus sequence of (hb)-C-X1-C-X2-(S/T)-X3-P-(hb)-C-D-X2-H, where hb represents a hydrophobic residue; we term this a CCCH-type [2Fe-2S] binding motif. The nine conserved residues in the motif contribute to iron ligation and structure stabilization. UV-visible absorption spectra indicated that mitoNEET can exist in oxidized and reduced states. Our study suggests an electron transfer function for mitoNEET and for other proteins containing the CCCH motif.2Fe-2S ͉ thiazolidinediones ͉ mitochondria T ype 2 diabetes is a growing global health problem characterized by insulin resistance and pancreatic ␤-cell dysfunction (1). Thiazolidinediones (TZDs) are a class of insulinsensitizing drugs used for treatment of type 2 diabetes (2), which includes rosiglitazone and pioglitazone currently in clinic use. The mechanism of action of TZDs has been generally attributed to their direct activation of peroxisome proliferator-activated receptor-␥, a ligand-binding nuclear receptor important for adipocyte differentiation and glucose homeostasis (3). However, accumulating evidence suggests that TZDs may also exert effects via a peroxisome proliferator-activated receptor-␥-independent pathway, particularly through modulation of mitochondrial activity (4-6). Colca et al. (7) have recently identified a protein in the mitochondrial membrane that crosslinks with photo-affinitylabeled pioglitazone (7). The crosslink could be competed by unlabeled pioglitazone, suggesting specificity in TZD binding. The protein was named mitoNEET because of its mitochondrial location and because of the presence of the sequence motif Asn-Glu-Glu-Thr (''NEET''). MitoNEET has a putative Nterminal transmembrane helix, which likely serves as a membrane anchor, and several invariant cysteine and histidine residues, which suggests that mitoNEET contains a CDGSH-type zinc finger (Fig. 1). However, its sequence is not homologous to any protein or domain of known function. Elucidation of the function and structure of mitoNEET is important to reveal its biological activity, to understand the pharmacology of TZDs, and to aid in the design of more potent antidiabetic drugs. Here, we show by structural characterization that mitoNEET is a previously unrecognized iron-sulfu...

Facile transfer of [2Fe-2S] clusters from the diabetes drug target mitoNEET to an apo-acceptor protein

“…These observations are consistent with the localization of the FeS cluster biogenesis machinery and key FeS protein metabolic functions in mitochondria (16,23). Moreover, as mitochondria are the primary energy providers of mammalian cells and key players in a large variety of metabolic processes (25), they have been implicated in metabolic diseases such as type II diabetes (26-29).Human mNT is composed of two protomers intertwined to form a unique structure with two domains; the β-cap and the cluster binding domain and is the founding member of the NEET fold (6,13,14,30). A single-coordinating histidine (H87) along with three cysteine ligands (C72, C74, C83) bind the [2Fe-2S] cluster and the single histidine has been shown to effect cluster redox and stability properties (6)(7)(8)(30)(31)(32)(33).…”

“…Human mNT is composed of two protomers intertwined to form a unique structure with two domains; the β-cap and the cluster binding domain and is the founding member of the NEET fold (6,13,14,30). A single-coordinating histidine (H87) along with three cysteine ligands (C72, C74, C83) bind the [2Fe-2S] cluster and the single histidine has been shown to effect cluster redox and stability properties (6)(7)(8)(30)(31)(32)(33).…”

“…Miner1 has recently been linked to autophagy, apoptosis, and aging (10, 11), and mis-splicing is associated with a rare disease, known as Wolfram Syndrome 2 (12). Together, mNT and Miner1 represent a new class of NEET iron-sulfur (FeS) proteins characterized structurally by their unique homodimeric fold and rare 3-Cys-1-His [2Fe-2S] cluster ligand environment (6,9,13,14).…”

“…Miner1 has recently been linked to autophagy, apoptosis, and aging (10, 11), and mis-splicing is associated with a rare disease, known as Wolfram Syndrome 2 (12). Together, mNT and Miner1 represent a new class of NEET iron-sulfur (FeS) proteins characterized structurally by their unique homodimeric fold and rare 3-Cys-1-His [2Fe-2S] cluster ligand environment (6,9,13,14).Iron-sulfur (FeS) cluster-containing proteins are key players in many essential processes, such as photosynthesis, respiration, and nitrogen fixation (15, 16). They appear in various compositions and confer upon FeS proteins the ability to accept or donate single electrons and/or iron, catalyze enzymatic reactions, or even function as regulatory proteins (17, 18).…”

Allosteric control in a metalloprotein dramatically alters function

“…They play critical roles as electron transfer proteins in processes such as photosynthesis, cellular respiration, nitrogen fixation (7,8), and catalysis (1). The newest member of the FeS cluster protein family, mitoNEET, is a uniquely folded homodimeric [2Fe-2S] protein, with each monomer bearing a single [2Fe-2S] cluster (9)(10)(11) (Fig. 1A).…”