JBIC Journal of Biological Inorganic Chemistry https://doi.org/10.1007/s00775-018-1538-8 MINIREVIEW The unique fold and lability of the [2Fe‑2S] clusters of NEET proteins mediate their key functions in health and disease Ola Karmi1 · Henri‑Baptiste Marjault1 · Luca Pesce2,3 · Paolo Carloni2,3 · Jose’ N. Onuchic4,5 · Patricia A. Jennings6 · Ron Mittler7 · Rachel Nechushtai1 Received: 4 December 2017 / Accepted: 26 January 2018 © The Author(s) 2018. This article is an open access publication Abstract NEET proteins comprise a new class of [2Fe-2S] cluster proteins. In human, three genes encode for NEET proteins: cisd1 encodes mitoNEET (mNT), cisd2 encodes the Nutrient-deprivation autophagy factor-1 (NAF-1) and cisd3 encodes MiNT (Miner2). These recently discovered proteins play key roles in many processes related to normal metabolism and disease. Indeed, NEET proteins are involved in iron, Fe-S, and reactive oxygen homeostasis in cells and play an important role in regulating apoptosis and autophagy. mNT and NAF-1 are homodimeric and reside on the outer mitochondrial mem- brane. NAF-1 also resides in the membranes of the ER associated mitochondrial membranes (MAM) and the ER. MiNT is a monomer with distinct asymmetry in the molecular surfaces surrounding the clusters. Unlike its paralogs mNT and NAF-1, it resides within the mitochondria. NAF-1 and mNT share similar backbone folds to the plant homodimeric NEET protein (At-NEET), while MiNT’s backbone fold resembles a bacterial MiNT protein. Despite the variation of amino acid com- position among these proteins, all NEET proteins retained their unique CDGSH domain harboring their unique 3Cys:1His [2Fe-2S] cluster coordination through evolution. The coordinating exposed His was shown to convey the lability to the NEET proteins’ [2Fe-2S] clusters. In this minireview, we discuss the NEET fold and its structural elements. Special attention is given to the unique lability of the NEETs’ [2Fe-2S] cluster and the implication of the latter to the NEET proteins’ cellular and systemic function in health and disease. Graphical abstract Ola Karmi, Henri-Baptiste Marjault and Luca Pesce contributed equally to this article. * Rachel Nechushtai [email protected] Extended author information available on the last page of the article Vol.:(0123456789)1 3 JBIC Journal of Biological Inorganic Chemistry Keywords [2Fe-2S] · Iron-sulfur clusters · Cisd(1–3) encoded NEET proteins · NEET-fold · NEET-cluster lability Preface composed of 127 amino acids and is encoded by the cisd3 gene. NAF-1 was identifed for its role in longevity [16] Iron-sulfur (Fe-S) proteins play a crucial role in a wide array as well as for its association with several human diseases, of biological processes including nitrogen fxation, photo- neuronal development and the basic cellular processes of synthesis and respiration [1–4]. These proteins are well char- autophagy and apoptosis [13, 15, 17–24]. All three NEET acterized as electron transfer proteins [5]. However, in recent proteins share a 39 amino acid sequence called the CDGSH years, evidence for additional functions such as sensors of domain (Fig. 1) [25]. The CDGSH domain contains a novel iron or oxygen [6, 7], enzymes [4], and gene expression fngerprint motif, the 3Cys:1His cluster coordination motif regulation [8] were attributed to Fe-S proteins. In addition, of the [2Fe-2S] cluster domain which characterizes the in recent years, an increased number of human diseases were NEET proteins [15, 26]. The human NEET proteins have found to be associated with dysfunctions of the Fe-S cluster all been shown to be associated with mitochondria; MiNT biogenesis pathway [8–11]. co-localizes with mitochondria while mNT and NAF-1 are Recently, a new class of [2Fe-2S] proteins, the NEET located on the outer mitochondrial membrane (OMM) [15, protein family, was discovered [12–14]. The frst member 27]. The major parts of mNT and NAF-1 face the cytosol of this family to be identifed was a mitochondrial protein and a single transmembrane helix at their N-terminal region mitoNEET (mNT) that binds the anti-type 2 diabetes drug anchors each monomer of these homodimeric proteins to pioglitazone. mNT is composed of 108 amino acids and the OMM [25, 28, 29]. NAF-1 was also found on the ER- is encoded by the cisd1 gene [12]. The name of mNT and mitochondrial associated membranes (MAM) that connects then of the entire NEET protein family is derived from the the ER to the OMM, as well as to the ER [13, 27]. There is C-terminal sequence Asn-Glu-Glu-Thr (NEET) of mNT a high similarity between the diferent NEET proteins. In [12]. In a subsequent study [15] two additional members humans, mNT and NAF-1 share about 54% identical and of the human NEET protein family were identifed. These 69% similar residues. In contrast, human MiNT shares about were the Nutrient-deprivation autophagy factor-1 (NAF- 50% identical and 63% similar residues with mNT, however, 1; previously Miner1) which is composed of 135 amino it has 38% identical and 50% similar residues to NAF-1 [30]. acids and is encoded by the cisd2 gene, and Mitochondrial Phylogenetic analysis of NEET proteins indicates that inner NEET protein (MiNT; previously Miner2) which is the CDGSH domain has been conserved throughout the 25-40 61-76 CDGSH CDGSH Bact. MiNT Trans- 1-79 membrane In-organelle Domain 74-89 CDGSH Cytosolic At-NEET 1-9 9-20 20-108 Trans-membrane In-organelle Domain 72-87 CDGSH Cytosolic mNT 1-13 13-31 31-108 In-organelle Trans-membrane 99-114 Domain CDGSH Cytosolic NAF-1 38-60 60-135 N-terminal 1-38 C-terminal Fig. 1 NEET proteins CDGSH organization. The location of the domains: in-organelle domain (checker texture), inter-membrane CDGSH domain(s) is shown in (red box) bacterial MiNT (blue), domain (diagonal lines pattern) and cytosolic domain (full color). At-NEET (green), mitoNEET (red) and NAF-1 (brown). Difer- The sequence interval is reported for each domain. The diferent ent textures of the boxes were used to distinguish between diferent regions specifed here are based on the sequence of each protein 1 3 JBIC Journal of Biological Inorganic Chemistry evolution of the NEET family. It is present in archaea The unique ‘NEET‑fold’ and structure and bacteria, mostly as monomeric proteins with two CDGSH domains [14, 31]. It has been suggested that the The unique ‘NEET-fold’ [25] is highly conserved from bac- gene duplication that resulted in the eventual formation teria through plants and humans NEET proteins [14, 30]. of mNT and NAF-1 in humans occurred around the time This fold and the NEET structures are unique compared to when vertebrates began to appear on Earth [31]. Further- the 132,017 structures that have been deposited to-date, out more, the closest CISD proteins to the ancient archetype of which 575 are known [2Fe-2S] proteins (http://www.rcsb. of eukaryotic NEET proteins was proposed to be similar org) [25, 33] (Fig. 2). to NEET proteins of the slime mold Dictyostelium dis- To understand the physiological role of each NEET coideum [31]. Since CISD proteins from snail, lancelet, protein structure it is crucial to know the diferences and hydra, lingual, sponge and sea anemone are more closely similarities between each family member. Human mNT and related to NAF-1, than to mNT, it has been suggested that NAF-1 as well as the plant At-NEET cytosolic structures NAF-1 evolved before vertebrates emerged and that mNT have been well characterized [25, 28, 29, 34]. The MiNT appeared via gene duplication after the radiation of ver- structure we refer to is that of the Magnetospirillum magne- tebrates [31]. In addition, some organisms lost specifc ticum bacterial homologue [14]. In this review, we used this classes of CISD proteins, such as plants that do not contain structure for the comparison of the diferent NEET proteins, MiNT-type NEET proteins. The CISD protein in plants, although the structure of human MiNT protein was pub- At-NEET (108 amino acids in length) (Fig. 1), resides lished recently [35]. In contrast to the homodimeric proteins both in chloroplast and mitochondria. At-NEET has a key mNT and NAF-1, MiNT/Miner2 is a monomeric protein role in plant development, senescence, reactive oxygen with two CDGSH domains (Figs. 1, 2) [14, 31, 35]. In mNT homeostasis, iron metabolism and homeostasis in diferent and NAF-1, each monomer contains a CDGSH domain, a cells [30–32]. At-NEET encoded by the (At5g51720) gene trans-membrane helix and an in-organelle domain (Fig. 1). shows 50 and 57% similarity to mNT and NAF-1, respec- Since for the homodimeric NEET proteins only the soluble tively, while its [2Fe-2S] binding domain has sequence domains were crystalized, our structural comparison relates identity to mNT and NAF-1 of about 75 and 88%, respec- to the available structures (Fig. 2) [25, 28–30, 36–38]. tively [30, 32]. The present mini-review aims to emphasize the molec- The ‘NEET fold’ ular components that contribute unique biophysical and biochemical properties to the NEET proteins. In particu- All NEET proteins, including the bacterial monomeric lar, we describe two properties of the NEET proteins that MiNT that folds into a two-fold pseudo symmetric struc- afect their function, the unique ‘NEET fold’ and the struc- ture, are comprised of two main domains: a β-cap domain tural elements of NEET proteins that determine the degree and a cluster binding domain (Fig. 2) [14, 25, 28, 29, 36, of liability of their [2Fe-2S] clusters. The implications of 37]. In the monomeric MiNT the β-cap domain comprises the latter in health and disease are also discussed.