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Anticancer and antibacterial activity in vitro evaluation of iridium(III) polypyridyl complexes.
JBIC Journal of Biological Inorganic Chemistry (2018) 23:599–612
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 / Published online: 12 February 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 membrane. 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 composition 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
rachel@mail.huji.ac.il
Extended author information available on the last page of the article
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JBIC Journal of Biological Inorganic Chemistry (2018) 23:599–612
Keywords [2Fe-2S] · Iron-sulfur clusters · Cisd(1–3) encoded NEET proteins · NEET-fold · NEET-cluster lability
Preface
Iron-sulfur (Fe-S) proteins play a crucial role in a wide array
of biological processes including nitrogen fixation, photosynthesis and respiration [1–4]. These proteins are well characterized as electron transfer proteins [5]. However, in recent
years, evidence for additional functions such as sensors of
iron or oxygen [6, 7], enzymes [4], and gene expression
regulation [8] were attributed to Fe-S proteins. In addition,
in recent years, an increased number of human diseases were
found to be associated with dysfunctions of the Fe-S cluster
biogenesis pathway [8–11].
Recently, a new class of [2Fe-2S] proteins, the NEET
protein family, was discovered [12–14]. The first member
of this family to be identified was a mitochondrial protein
mitoNEET (mNT) that binds the anti-type 2 diabetes drug
pioglitazone. mNT is composed of 108 amino acids and
is encoded by the cisd1 gene [12]. The name of mNT and
then of the entire NEET protein family is derived from the
C-terminal sequence Asn-Glu-Glu-Thr (NEET) of mNT
[12]. In a subsequent study [15] two additional members
of the human NEET protein family were identified. These
were the Nutrient-deprivation autophagy factor-1 (NAF1; previously Miner1) which is composed of 135 amino
acids and is encoded by the cisd2 gene, and Mitochondrial
inner NEET protein (MiNT; previously Miner2) which is
composed of 127 amino acids and is encoded by the cisd3
gene. NAF-1 was identified for its role in longevity [16]
as well as for its association with several human diseases,
neuronal development and the basic cellular processes of
autophagy and apoptosis [13, 15, 17–24]. All three NEET
proteins share a 39 amino acid sequence called the CDGSH
domain (Fig. 1) [25]. The CDGSH domain contains a novel
fingerprint motif, the 3Cys:1His cluster coordination motif
of the [2Fe-2S] cluster domain which characterizes the
NEET proteins [15, 26]. The human NEET proteins have
all been shown to be associated with mitochondria; MiNT
co-localizes with mitochondria while mNT and NAF-1 are
located on the outer mitochondrial membrane (OMM) [15,
27]. The major parts of mNT and NAF-1 face the cytosol
and a single transmembrane helix at their N-terminal region
anchors each monomer of these homodimeric proteins to
the OMM [25, 28, 29]. NAF-1 was also found on the ERmitochondrial associated membranes (MAM) that connects
the ER to the OMM, as well as to the ER [13, 27]. There is
a high similarity between the different NEET proteins. In
humans, mNT and NAF-1 share about 54% identical and
69% similar residues. In contrast, human MiNT shares about
50% identical and 63% similar residues with mNT, however,
it has 38% identical and 50% similar residues to NAF-1 [30].
Phylogenetic analysis of NEET proteins indicates that
the CDGSH domain has been conserved throughout the
61-76
25-40
CDGSH
Transmembrane
In-organelle Domain
CDGSH
74-89
Cytosolic
1-9
9-20
In-organelle
Trans-membrane
Domain
In-organelle
13-31
1-38
Fig. 1 NEET proteins CDGSH organization. The location of the
CDGSH domain(s) is shown in (red box) bacterial MiNT (blue),
At-NEET (green), mitoNEET (red) and NAF-1 (brown). Different textures of the boxes were used to distinguish between different
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CDGSH
mNT
31-108
99-114
CDGSH
Cytosolic
N-terminal
At-NEET
72-87
Trans-membrane
Domain
38-60
CDGSH
20-108
Cytosolic
1-13
Bact. MiNT
1-79
60-135
NAF-1
C-terminal
domains: in-organelle domain (checker texture), inter-membrane
domain (diagonal lines pattern) and cytosolic domain (full color).
The sequence interval is reported for each domain. The different
regions specified here are based on the sequence of each protein
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evolution of the NEET family. It is present in archaea
and bacteria, mostly as monomeric proteins with two
CDGSH domains [14, 31]. It has been suggested that the
gene duplication that resulted in the eventual formation
of mNT and NAF-1 in humans occurred around the time
when vertebrates began to appear on Earth [31]. Furthermore, the closest CISD proteins to the ancient archetype
of eukaryotic NEET proteins was proposed to be similar
to NEET proteins of the slime mold Dictyostelium discoideum [31]. Since CISD proteins from snail, lancelet,
hydra, lingual, sponge and sea anemone are more closely
related to NAF-1, than to mNT, it has been suggested that
NAF-1 evolved before vertebrates emerged and that mNT
appeared via gene duplication after the radiation of vertebrates [31]. In addition, some organisms lost specific
classes of CISD proteins, such as plants that do not contain
MiNT-type NEET proteins. The CISD protein in plants,
At-NEET (108 amino acids in length) (Fig. 1), resides
both in chloroplast and mitochondria. At-NEET has a key
role in plant development, senescence, reactive oxygen
homeostasis, iron metabolism and homeostasis in different
cells [30–32]. At-NEET encoded by the (At5g51720) gene
shows 50 and 57% similarity to mNT and NAF-1, respectively, while its [2Fe-2S] binding domain has sequence
identity to mNT and NAF-1 of about 75 and 88%, respectively [30, 32].
The present mini-review aims to emphasize the molecular components that contribute unique biophysical and
biochemical properties to the NEET proteins. In particular, we describe two properties of the NEET proteins that
affect their function, the unique ‘NEET fold’ and the structural elements of NEET proteins that determine the degree
of liability of their [2Fe-2S] clusters. The implications of
the latter in health and disease are also discussed.
The unique ‘NEET‑fold’ and structure
Fig. 2 NEET proteins’ structures solved by X-ray crystallography.
Structure of monomeric bacterial MiNT (blue colored; PDB-ID: 3tbn
[14]), and dimeric (monomers A are reported with lighter colors) AtNEET (green, 3s2q [30]), mitoNEET (red, 2qh7 [15]) and NAF-1
(brown, 4oo7 [38]) proteins, and their superposition. The [2Fe-2S]
cluster atoms are shown in orange-yellow spheres. The superposi-
tion of the four proteins shows the high structural similarity shared
between the NEET proteins. The crystallized part of the homodimeric
NEET proteins is limited to the cytosolic domain (fully colored part
in Fig. 1) without the linkers to the membrane and the intra-organelle
parts
The unique ‘NEET-fold’ [25] is highly conserved from bacteria through plants and humans NEET proteins [14, 30].
This fold and the NEET structures are unique compared to
the 132,017 structures that have been deposited to-date, out
of which 575 are known [2Fe-2S] proteins (http://www.rcsb.
org) [25, 33] (Fig. 2).
To understand the physiological role of each NEET
protein structure it is crucial to know the differences and
similarities between each family member. Human mNT and
NAF-1 as well as the plant At-NEET cytosolic structures
have been well characterized [25, 28, 29, 34]. The MiNT
structure we refer to is that of the Magnetospirillum magneticum bacterial homologue [14]. In this review, we used this
structure for the comparison of the different NEET proteins,
although the structure of human MiNT protein was published recently [35]. In contrast to the homodimeric proteins
mNT and NAF-1, MiNT/Miner2 is a monomeric protein
with two CDGSH domains (Figs. 1, 2) [14, 31, 35]. In mNT
and NAF-1, each monomer contains a CDGSH domain, a
trans-membrane helix and an in-organelle domain (Fig. 1).
Since for the homodimeric NEET proteins only the soluble
domains were crystalized, our structural comparison relates
to the available structures (Fig. 2) [25, 28–30, 36–38].
The ‘NEET fold’
All NEET proteins, including the bacterial monomeric
MiNT that folds into a two-fold pseudo symmetric structure, are comprised of two main domains: a β-cap domain
and a cluster binding domain (Fig. 2) [14, 25, 28, 29, 36,
37]. In the monomeric MiNT the β-cap domain comprises
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four β-strands. The β1 strand pairs with β4 forming a two
stranded antiparallel sheet (see Table 1), while β2 pairs
with β3 to form a second two stranded antiparallel sheet.
These two β-sheets pack against each other and are linked
by two loops between β1 and β2 (L2) and between β3 and
β4 (L4). These two sheets comprise the β-cap domain.
The cluster binding domains of MiNT are distinct and are
characterized by three regions (L1, L3 and L5). The L3
loop is connected to the first [2Fe-2S] CDGSH coordination motif in the N-terminal (Cys25, Cys27, Cys36 and
His40), and the L5 loop-helix is connected to the second
[2Fe-2S] CDGSH motif in the C-terminal (Cys61, Cys63,
Cys72 and His76) [14]. These two structural halves are
connected by both hydrophobic and polar interactions.
However, the backbone fold with its pseudo twofold symmetry is remarkably similar in all eukaryotic homodimeric
NEET proteins (see Fig. 2 for the crystallized structures
of the proteins; their sequences are contained in the fully
colored part of Fig. 1) [14]. The backbone structures of
the β-cap domains of the three eukaryotic NEET proteins
(At-NEET, mNT and NAF-1) are highly similar [30]. In all
three proteins, the β-cap domain primarily comprises three
long β-strands per monomer (each containing 28 amino
acids) [25, 29]. These β-strands are assembled in two symmetric β-sheets, which in each monomer are composed of
two antiparallel strands from one monomer and a third
parallel swapped-strand from the other monomer. The two
β-sheets and the two linking loops on the top of the sheets
(L2 in both monomers) form the β-cap sandwich domain
(see Table 1 and see Fig. 2: The superposition comparison)
[25, 29, 30]. The β-cap domain is held across from L2
by the cluster binding domain (Fig. 2). The cluster binding domain of each monomer contains a CDGSH domain
(one per monomer), followed by an α-helix structure (see
Table 1). The N-terminal of the soluble domain (L1) and
the loop connecting the α-helix to the β-cap (L4) belong
to the cluster binding domain. The structure of the N-terminus of NEET proteins, connected to the trans-membrane
helices, is not yet known. However, the crystallographic
structure of mNT showed that the cytoplasmic tethering
domain could assume different orientations. This suggests
high flexibility which may participate in protein–protein
interaction [37], as well as affect the coupling between
the folding and dimerization of NEET proteins [39, 40].
Despite the very high level of similarity in the backbone
structures, differences do exist between the AT-NEET,
NAF-1 and mNT structures. For example, At-NEET and
NAF-1 are slightly wider on the top of the loop connecting the intertwined strand to the other monomer in the
β-cap domain, due to the presence of an extra amino acid
in L2 that is not present in mNT (Asn69 in At-NEET and
Thr94 in NAF-1) [29, 30]. In the crystalized structure of
NAF-1, there is one free non-conserved Cys92 located to
the upper part of the β-cap domain that was replaced with
the isosteric Ser (C92S), due to instability and aggregation
problems in the purification process [29].
Table 1 The amino acids constituting the secondary structures elements and the protein domains of the NEET proteins
Protein
Cluster binding
β-Cap
Bact. MiNT
At-NEET
mNT
NAF-1
1–12, 25–48, 61–79
44–59, 74–103
42–58, 72–101
68–84, 99–128
13–24, 49–60
60–73, 104–108
59–71, 102–108
85–98, 129–135
Bact. MiNT
At-NEET
mNT
NAF-1
Bact. MiNT
At-NEET
mNT
NAF-1
β1
β2
β3
β4
α1
13–16
60–63
59–62
85–88
23–24
70–73
68–71
95–98
49–50
104–107
102–105
129–132
57–60
–
–
–
–
89–96
86–94
113–121
Cys1
Cys2
Cys3
His
25,61
74
72
99
27,63
76
74
101
36,72
85
83
110
40,67
89
87
114
The table details the amino acid indexes comprising the NEET cluster binding and β-cap domains (top panel); β-strands and α-helix of a single monomer (middle panel); and the coordinating residues of the [2Fe-2S] clusters (low panel). The C
ysi indexing follow the sequence of the
conserved CDGSH domain. In MiNT the values of the two CDGSH domains of the protein are indicated. One should note that the amino acids
relate to the structure of different NEET proteins solved by X-ray crystallography: At-NEET, 3s2q [30]; mitoNEET, 2qh7 [15]; NAF-1, 4oo7
[38]; bacterial MiNT, 3tbn [30]
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Differences in the repartition of the hydrophobic/
charged residues in the homodimeric NEET
structures
The NEET proteins that are homodimers are stabilized by
repartition of hydrophobic and charged residues [25, 30, 39].
Even though the distribution of the hydrophobic residues
appears similar in all homodimeric NEET proteins, there
are differences between the members [14, 29, 30]. On the
surface of the mNT structure there is a convex hydrophobic
ring that does not exist in At-NEET and NAF-1. This is
composed of two Phe residues co-localized near the conserved Tyr. In contrast, At-NEET and NAF-1 have different
hydrophobic residues co-localized to the Tyr which create a
hydrophobic cleft in the same domain [30, 37] (see Fig. 3).
The localization of Tyr is similar in all of the NEET proteins,
when comparing root mean square displacement (RMSD)
of the hydroxybenzyl group of Tyr, however, the level of
similarity is highest between NAF-1 and At-NEET. There
is a RMSD of 1.1 Å between NAF-1 (Tyr98) and At-NEET
(Tyr73), whereas a RMSD of 1.5 Å exists between mNT
(Tyr71) and either NAF-1 or At-NEET.
In general, the charged residues are distributed at the top
of the β-cap domain and on the cluster-binding domain surface (see Fig. 4). This repartition of charged residues separated by the hydrophobic core (described above) leads to
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polarized/charged domains (depending on the family member) at the top and at the bottom of the two main domains
of the proteins. In the folded part of the cytosolic domain of
NEET proteins (residues 43–108 in mNT, 69–135 in NAF-1
and 44–110 in At-NEET), mNT is neutral, there is no net
charge in electron units at pH 7.0, whereas NAF-1 and AtNEET both have a net positive charge (~ + 2 at pH 7.0)
[41]. The electrostatics residues (marked onto the overlaid
structures in Fig. 4) provide an insight to this change [14,
25, 29, 30].
Taken together, the differences described above for the
structures and hydrophobic/electrostatic residues (Figs. 2,
3, 4), are associated with the variability in the homodimeric
packaging, the amino acid composition and side chain orientation. For example, the mNT-Arg73 side chain, near the
[2Fe-2S] cluster, forms an internal inter-monomer hydrogen bond with His58 side chain. The arginine is highly conserved across all the homodimeric NEET proteins (Arg100
inNAF-1 and Arg75 in At-NEET) [14, 30], while His 58
mNT is not conserved in NAF-1 and At-NEET. Intriguingly these Arg residues bind to the side chain of Asn84
and Asp59 of NAF-1 and At-NEET, respectively [29, 30].
This kind of difference in inter-monomer interactions can
lead to differences in the stability of the dimeric structure
among the NEET proteins. They may also affect different
interactions of the NEET proteins and their partners and
Fig. 3 Central hydrophobic
domains of the NEET proteins.
The amino acids belonging to
the hydrophobic central patch of
mNT (red), NAF-1 (orange) and
At-NEET (green) are shown in
ball and stick representations
over the structures of mNT
[25], NAF-1 [38] and At-NEET
[30] structures colored in grey
shades from the brighter to
the darker, respectively. The
localization of the conserved
Tyr is affected by the displacement of the surrounding amino
acids, modulating, therefore, its
position and orientation
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40
20
mNT
At-NEET
NAF-1
90deg
90deg
0
-20
-40
E le ctrostatic potential [m V ]
60
-60
Fig. 4 Electrostatic potential on the NEET protein’s surface. The
electrostatic potential values (estimated using NEET proteins’ force
files [85] and APBS electrostatic [103]) of mNT [25], NAF-1 [38]
and At-NEET [30] are here reported over each protein surface. The
side facing the plane of the β-sheet (top) and the side view (bottom)
are here reported. The color code refers to the electrostatic potential
values reported on the right
may also affect the lability/stability/redox potential of the
[2Fe-2S] clusters [29].
The [2Fe-2S] cluster coordinating His is located at the
N-terminus of the α-helix within the cluster-binding domain.
It is solvent accessible and it coordinates the outermost Fe of
the [2Fe-2S] with one of the three Cys-ligands (see Fig. 5).
The lability of the NEET [2Fe-2S] cluster is largely attributed to this residue (see the lability of the NEET cluster
section, below). The last two Cys ligands coordinating the
innermost iron of the [2Fe-2S] are buried inside the structure
(see Fig. 5) [25, 29, 39].
The cluster binding domain
The cluster binding domain, which is part of the CDGSH
domain in all NEET proteins, harbors the [2Fe-2S] cluster.
In general, the cluster binding domain is similar across species, with a higher sequence similarity compared to the other
domains of the proteins and it is composed of the unique
coordination of 3Cys:1His. The two [2Fe-2S] clusters interact by inter-cluster dipolar coupling [42, 43]. These clusters
of the NEET proteins were shown to be redox-active. The
redox properties can be tuned upon changes in the surrounding environment of the protein [26, 37, 44, 45]. Moreover,
the [2Fe-2S] clusters may communicate via inter-dimer electron transfer, even when the clusters are at different oxidation states [43].
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The labile [2Fe‑2S] clusters of NEET proteins
Different biophysical and biochemical methodologies were
used for the characterization of the [2Fe-2S] clusters of
NEET proteins [26, 42–62]. These included UV–Vis absorption spectroscopy, mass spectroscopy (MS) [26], electron
paramagnetic resonance (EPR) [42] and resonance Raman
[45]. When the structure of the NEET proteins became
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Fig. 5 [2Fe-2S] cluster-binding domain of NEET proteins. Comparison of the superposition of the cluster-binding domain using the same
color-code as in Fig. 2. The details of the superposition of the [2Fe2S] 3Cys:1His pocket of each protein is shown within the box. In the
left hand side figure the overlap between the proteins is not ideal for
MiNT. Nevertheless, the similarity of the inner coordination sphere
of ligands of the different NEET proteins is high
available, it provided molecular–atomic explanations to the
different biophysical measurements. For example, the MS
of holo-mNT (mNT with the cofactor) vs. that of apo-mNT
(mNT without the cofactor), obtained by lowering the pH,
showed a molecular weight of 9230.6 (± 0.2) Da per hohomNT monomer and of 9056.9 (± 0.2) Da per apo-mNT
monomer [26]. This indicated that the difference between
the holo- and apo- forms of mNT is 173.7 (± 0.3) Da [26],
which corresponds unambiguously to a [2Fe-2S] cluster.
Indeed X-ray structures of the NEET proteins confirmed
these findings. Moreover, biophysical studies made it possible to characterize the implications of the pH effects on the
NEET proteins’ labile metal center [26].
The fingerprint absorption peak of the NEET proteins’
cluster in its oxidized form was found to be 458 nm; upon
reduction this peak absorption is highly decreased [26, 39].
The ~ 90% decrease in the 458 nm absorption, under reducing conditions, can be fully recovered by exposing the NEET
proteins to oxygen, proving that the [2Fe-2S] cluster of the
NEET proteins is redox-active [7, 20, 26]. For a detailed
description of the biophysical properties of the NEET cluster
please refer to our previous review published by Tamir and
his coworkers in BBA review—Biochimica et Biophysica
Acta (BBA)-Molecular Cell Research [39].
We focus here on the molecular determinants of
NEET proteins that contribute to the unique lability
of their [2Fe-2S] clusters. The [2Fe-2S] cluster of all
known NEET proteins are coordinated by 3Cys:1His.
This feature distinguishes them from the highly abundant
4Cys coordinating structure of for example Ferredoxin,
or the 2Cys:2His coordination of Rieske [39]. When first
reported, this NEET protein’s coordination was unique
among Fe-S proteins. In years to follow, e.g. in a D38A
mutant of the iron-sulfur scaffolding protein IscU it was
shown that the [2Fe-2S] cluster composed on the scaffold
protein is also coordinated by 3Cys:1His [63]. Yet, crystal structure of the system indicated that while in NEET
proteins the His coordinates the cluster via N
δ, in the IscU
protein the cluster is coordinated by the N
ε of the His [64].
When the structures of mNT and NAF-1 became available [25, 28, 29, 36, 37], the [2Fe-2S] clusters coordinating
residues for mNT and NAF-1 were identified as Cys72/99,
Cys74/101, Cys83/110 and His87/114, respectively [26,
29] (see Table 1, which also includes the plant At-NEET
and bacterial-MiNT cluster coordinating residues). As
Fig. 5 indicates, the superposition of the [2Fe-2S] cluster
coordination sites of bacterial-MiNT through plant AtNEET and human mNT and NAF-1, form a nearly perfect
overlay. The latter indicates that the cluster coordination
site of NEET proteins was preserved through evolution
from bacteria to human, which supports similar functional
roles for the NEET proteins in all organisms. Importantly,
differences in amino acid composition also create opportunities for selectivity in protein binding partners providing overlapping but not identical functions for multiple
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paralogs within a single organism (e.g., the cluster transferability between mNT or NAF-1 to anamorsin [55]).
The finding that NEET proteins have as a fourth coordinating cluster ligand, the His residue, which induces a pHdependent-lability to their cluster, was proven as a unique
feature of their [2Fe-2S] clusters. Indeed, the latter does not
exist in Ferredoxins’ [2Fe-2S] (4Cys coordination) which
have a high level of cluster stability under similar buffer
conditions [26, 39, 45]. Moreover, when the coordinating
His was replaced with a Cys, (H87C, H114C and H89C in
mNT, NAF-1 and At-NEET, respectively), the [2Fe-2S]
clusters of the NEET proteins were stabilized, similar to
that of the Ferredoxin cluster. This stability is maintained
in acidic pH [20, 26, 29, 30, 38]. In addition, the lability of
the [2Fe-2S] cluster was shown to depend on the oxidation
state of the cluster itself, and when the [2Fe-2S] cluster is in
its reduced state in wild type His-containing NEET proteins,
it is stable even at low pH [50]. This property may suggest that one of the functional roles of NEET proteins is to
serve as a redox-sensing proteins [7]. Fe-S cluster containing
proteins have the ability to play a role as sensors by losing
their cluster, accommodating another type of cluster such
as switching between [4Fe-4S] and [2Fe-2S], or receiving/
transfering electrons, causing a change in the redox state of
the cluster [65]. This sensing mechanism controls the activity of the Fe-S proteins in response to redox signals, through
the changes of the redox state of their cluster [7]. Based on
the cluster lability/stability studies we have suggested that
NEET proteins are involved in ROS and Iron homeostasis
[22, 30, 39, 66]. Recently, NEET proteins were also suggested to belong to Fe-S proteins that have a mechanism
that when their [2Fe-2S] cluster are reduced the proteins are
considered to be in a “dormant” state [7]; and when the cluster receives a signal that induces its oxidations, the NEET
proteins are switched into an active state. The efficiency
of this sensing mechanism may help cells to turn on their
survival pathways quickly and recover from any stressful
conditions [7].
As stated above, the pH-dependent stability of the NEET
proteins’ [2Fe-2S] clusters, was associated with His protonation. Lowering the pH induced an accelerated loss of
the clusters and its half-life was significantly decreased [26,
29]. To investigate the role of His in more details, His was
replaced with Cys in mNT and NAF-1 proteins. Differences
were observed between the mutants and their respective WT.
Thus, the H114C-NAF-1 mutant structure shows the constant formation of a hydrogen bond between the Lys81 and
Asn115 [38] while the H87C-mNT mutant structure showed
two conformers having two distinct configurations for Lys55
and Cys87 [67].
This was also supported by the investigation of the residues surrounding His. In particular, the Lys that associates with mNT His87 (Lys55) plays an important role in
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conveying cluster lability, and the hydrogen bonding network helps to tune this stability, but not to affect the reduction potential [50]. The [2Fe-2S] cluster is also considered
to be redox active with an Em value of 0 mV (± 10 mV)
for mNT and NAF-1 at pH 7.5, the Em value is pH dependent and may decrease by approximately 50 mV per pH unit
when pH is being increased from 7.5 to 10. This supports a
mechanism whereby reduction is proton-coupled, and this
often has a relevance to function [29]. As reduction is coupled to proton uptake, redox titration indicated that at a pH
above the pKa of the oxidized state (pKox) and below the
pKa of the reduced state (pKred), the reduction gives an
uptake for a proton that is coupled to the His87 in mNT,
which results in a pH-dependence vibrational interaction
with the [2Fe-2S] center [47]. However, measurements of
the protein redox potential and protein film voltammetry for
the mNT [2Fe-2S] cluster were used to determine the pKa
of the protein [44, 47, 50]. These models give pKa results
of about 6.5 [44, 50] and 6.8 [47] for the oxidized [2Fe-2S]
cluster. These models introduce empirical parameters that do
not reveal the source of the proton donor and are not related
to a specific amino acid. For this reason, further work is
needed to investigate the protonation state of the coordinating His directly [68].
In addition to studies on the effects of pH on the clusterlability, reduction of the coordinating His and pKa, the His
to Cys mutations of the NEET cluster-coordination have also
been found to affect the cluster redox potential (Em). Em can
range from ~ 30 mV in wild type mNT/NAF-1 and about
0 mV in At-NEET, to ~ 10 times more negative values in
mutants (> − 300 mV) such values are closer to the cluster
Em of plant Ferredoxin (− 325 mV) and vertebrate Ferredoxin (− 235 to − 273 mV) [30, 39, 47, 69, 70].
Moreover, the variation between resonance Raman spectra of mNT protein and its Ferredoxin like mutant H87C
which is found within peaks in the region of 250–300 cm−1
[45], support the hypothesis that the energy required for
the cleavage of the Fe–N bond of a single His residue is
modulated within the physiological pH range [45]. This may
be considered as the first but not rate-limiting step prior to
cluster loss, and in addition, this may be critical for in vivo
functions of the NEET proteins [45]. This fact was further
confirmed experimentally by the EPR study [42].
Several recent studies are focused on characterizing the
electron transfer properties of NEET proteins and on the
binding of the NEET proteins/[2Fe-2S] clusters to other
small molecules. By mimicking the [2Fe-2S] harbor of
NEET proteins in a model system, proton coupled electron
transfer ability and the corresponding thermodynamic properties and function of the His ligand could be investigated.
Some studies focused on possible electron donors/acceptors for mNT [2Fe-2S] clusters in mitochondria such as flavin reductase which reduces flavin mononucleotides (FMN)
JBIC Journal of Biological Inorganic Chemistry (2018) 23:599–612
to FMNH2 using NADH as electron donor. It was shown that
mNT mediates the oxidation of NADH with concomitant
reduction of oxygen [60, 61]. Interestingly, it was also shown
that Fe-S clusters involved in Cys-coordination to protein
are disrupted by nitric oxide (NO) [58, 71]. However, when
the [2Fe-2S] clusters of MiNT are in a reduced state, MiNT
can bind NO without disrupting the cluster. In addition, the
other two human NEET proteins, mNT and NAF-1, fail to
bind NO, but a single mutation, (D96V in mNT, or D123V
in NAF-1) facilitates the binding of NO to the [2Fe-2S]
cluster. This indicates that subtle changes to these proteins
may switch their ability to bind NO, and thereby facilitate
signaling in cells and modulation of mitochondrial function
through NO signaling [58].
Despite the accumulation of valuable structural and
molecular information on the ‘NEET fold’ as well as information on the structural and labile nature of the [2Fe-2S]
clusters and how these affect NEET protein function, many
issues remain to be solved. One such enigma is why mutations of amino acids that are at a large distance (more than
20 Å) from the [2Fe-2S] cluster, e.g. in the β-cap, affect the
cluster properties (Em values, cluster transfer rates). Another
concerns how cluster loss affects the structure of NEET
protein. It was shown that cluster loss induces the unfolding of mNT [46, 52, 62]. However, nothing is known about
the unfolding pathways of the NEET proteins. It is widely
agreed that in the last two decades [72], molecular simulations have provided valuable insights into the structural
determinants, the electronic structure and the spectroscopic
properties of Fe-S proteins with [2Fe-2S] and [4Fe-4S]
centers [72–78]. We here describe how theoretical simulation assisted in understanding some un-solved issues of the
NEET proteins like the ones underlined above.
Theoretical studies on NEET proteins
Computational studies have shed insights on the complex
nature of the bonds between the Fe-S centers and the thiolated sulphurs of the Cys residues [72–84]. These studies
used a partial or full application at the quantum mechanical level (QM). Full QM studies are usually performed on
reduced domains of the protein or model systems representative of the region containing the cofactor. Since electronic
processes can be affected by environmental effects, e.g.
arising from the solvent and/or biomolecular frame, hybrid
methods, combining QM and molecular mechanics (MM)
allow for the electronic properties of the cofactor binding
site to be characterised. In particular simulation studies have
been extended to the bc1 protein complex [82, 83], that contains [2Fe-2S] clusters in which one of the two iron atoms
is coordinated by two 2His residues. In addition, a study
in which computational and experimental methods were
607
coupled, was carried out on a model system mimicking the
unique 3Cys:1His [2Fe-2S] cluster of NEET proteins. In
this study, it was shown that concerted proton- and electrontransfer is involved in the process of reduction/oxidation of
the [2Fe-2S] clusters [84].
Our team has recently applied established theoretical
tools to study the peculiar coordination 3Cys:1His of the
[2Fe-2S] cluster of the NEET proteins. The contribution of
the different amino acids in the cluster binding region or in
the distant β-cap domain to the clusters’ properties such as
lability and reduction potential were studied. In addition,
quantum mechanical calculations were applied to uncover
key factors for the Fe–N bond’s reactivity leading to cluster
liability [85].
Global structural information of mNT’s protein frame and
the effects of chemical/physical properties of mNT on large
time scales and spatial scales were uncovered [40, 51, 85,
86]. In particular computational studies on the mNT folding highlight the importance of the β-cap domain during
the folding of the protein [40, 86]. An analysis of coupled
regions on the folding landscape led us to predict where we
could allosterically control the cluster properties from afar.
This was followed by mutational and full structural analysis
(see Fig. 6a) [51]. Mutations in amino acids of the β-cap
domain affected the redox potential of the [2Fe-2S] cluster
of mNT, less than the other mutations in amino acids that
are proximal to the [2Fe-2S] cluster affect its redox potential [47, 51]. However, these mutations highly affected the
mNT [2Fe-2S] cluster stability and cluster transfer rates
[51]. Interestingly, cluster stability and cluster transfer rate
were not correlated. X-ray structural analysis of the mutant
proteins proved that the global fold of the protein remains
unchanged. But, using energy landscape theory and all-atom
structure based models, it was possible to understand that
dynamic twisting of the β-cap domain result in scissoring of
the distal cluster binding domain. The distal cluster binding
domain altered the dynamic motions and transient distances
between the coordinating His and the [2Fe-2S] cluster. Thus,
while the global fold is maintained, changes in dynamic
motions altered by mutations in sites that are 20 Å removed
from the cluster, regulate cluster functional properties [51].
All atoms molecular dynamics on the NEET protein
[2Fe-2S] cluster binding domain lead to two important
suggestions. First, that the sensitivity to pH environmental
variations [85] is mainly due to the differences between the
amino acid that follows the coordinating His. This affects the
localization of the conserved Lys (55 in mNT, 81 in NAF1), which shields the His:Nε from the solven. Second, that
conformational changes in mNT and NAF-1 are induced by
a single (see Fig. 6b) or a double cluster release [85]. Upon
the release of one cluster the α-helix of the monomer without the cluster is lost and, in addition, part of the structure
of the other monomer is also affected. In case of loss of both
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608
JBIC Journal of Biological Inorganic Chemistry (2018) 23:599–612
Fig. 6 mNT modifications and theoretical analysis. (a, left) Crystal
structure of mNT [25] highlighting the allosteric mutated residues
at the top of the β-cap (violet). All crystal structures are available of
the mutated proteins [51]. The β-cap mutations alter the coordinated
motions of the domain (a, center panel), correlated with the flexibility of the cluster binding domain (right) and, in particular, with
the coordinating histidine (yellow colored arrow) [51]. The colors of
the cartoon structure in the central and right panels span from blue
to red representing the movement along the principal vibrational
mode of the protein. (b, left) Representative structure of the mNT
in absence of one [2Fe-2S] cluster from monomer A obtained using
replica exchange molecular dynamics [52]. (b, right) The effects of
the cluster absence on standard deviation maps [52]. Here each pixel
represents the standard deviation of the distance between each residue
couple. The regions which are mostly affected by the cluster absence
are the α-helix of the monomer losing the cluster and the L1 domain
of the other [85]
the clusters, NEET proteins undergo a large structural rearrangement such as loss of both helices, along with the partial
loss of the β-sheet structures.
In conclusion, the computational studies add valuable
insight into questions raised by experimental results. Theoretical results also pose new questions for experimentalists.
We believe that coupling experimental and theoretical investigations of NEET proteins will lead to detailed mechanistic information on the structure–function relationships, in
particular with regard to the function of [2Fe-2S] cluster
lability. Another major area in which computational tools
are critical, is in clarifying the mode of binding of drugs
to NEET proteins. The discovery of the NEET family was
through mNT binding to pioglitazone [12], and more than a
decade latter computational docking analysis resolved how
pioglitazone stabilizes the [2Fe-2S] cluster [22]. The same
holds true for other families of small molecules [87]. The
drug design and binding studies are key for the pharmacological studies related to NEET proteins. Moreover, computational methodologies such as direct coupling analysis
are also critical for defining NEET-partner proteins, e.g.,
13
JBIC Journal of Biological Inorganic Chemistry (2018) 23:599–612
NAF-1-BCL-2 [21] and for clarifying the cellular pathways
that NEET proteins participates in.
NEET protein involvement in diseases
NEET proteins are important in health and diseases. In
healthy subjects, the cisd2 gene, encoding NAF-1 protein,
was shown to reside on chromosome 4. It is involved in
longevity [16], and in mice several studies indicated that
suppressed expression of cisd2 led to shortened life spans
[88]. In human pathologies mNT and NAF-1 were shown
to be involved in diabetes and obesity [89, 90], neurodegeneration and cardiovascular abnormalities, and skeletal
muscle maintenance [13, 29, 91], they were also implicated in autophagy, apoptosis [18, 21, 23, 92], aging [16,
93] and cancer [22, 23, 90, 94–97]. In addition, NEET
proteins are implicated in the rare genetic disease Wolfram
Syndrome 2 (WFS-2). In WFS-2 homozygous intragenic
missense mutations lead to exon skipping introducing an
early stop codon which results in the elimination of the
NAF-1 protein from cells [13, 98–100]. Another genotype,
leading to abnormal expression of NAF-1, was related to
WFS-2 [101]. The phenotype of this syndrome is associated with hearing deficiencies, neurodegeneration, sever
blindness, diabetes and a lower life expectancy [13, 99,
100].
The NEET proteins mNT and NAF-1 and recently
human MiNT were shown to be involved in iron/Fe-S/ROS
homeostasis in cells [23, 26, 30, 39, 49, 52, 55, 66]. These
proteins, and in particular mNT and NAF-1, were found to
function in the same pathways in mammalian cells [59].
By overexpressing mNT or NAF-1 in cells, activation of
apoptosis and/or autophagy was prevented while cellular
proliferation was supported by cellular resistance to oxidative stress [22, 96]. On the other hand, overexpression
of the NAF-1 variant (H114C) did not promote cellular
proliferation. In addition, such overexpression suppressed
xenograft tumor growth [22]. Suppressing mNT or NAF-1
expression, results in over-accumulation of mitochondrial
iron and ROS in mammalian cells, leading to the activation of autophagy and apoptosis [23, 39, 66]. This may be
mediated through the interaction with other proteins; such
as BCL-2 a key protein involved in autophagy/apoptosis
regulation which is known to interact with NAF-1 [18,
21]. The interaction of NAF-1 with BCL-2 is thought to be
controlled by the presence or absence of the [2Fe-2S] clusters of NAF-1 [18]. Since the presence or absence of the
cluster in the protein may have a functional role in cells,
it was important to evaluate the ability of the proteins to
donate or accept clusters. This hypothesis was confirmed
using different apo-accepters such as apo-Ferredoxin [20,
48]. By further investigating this ability it was critical to
609
find physiological candidates for accepting the [2Fe-2S]
cluster. The first one to be identified for both mNT and
NAF-1 was Anamorsin, which is an electron transfer protein and is required for cytosolic Fe-S cluster assembly
[55]. In addition, the mNT protein donates its clusters to
cytosolic Aconitase [52, 102]. This was confirmed using
the mutant forms of the protein H87C and H87S that
replace the His with other amino acids and stabilize the
cluster of the protein [102]. Another interaction between
NEET proteins and the cytosolic Fe-S protein assembly
machinery was through the redox switch mechanism of
their clusters, through their ability to control the cluster
transfer repair pathway, by transferring the NEET clusters to Anamorsin [57] and cytosolic Aconitase [52]. Most
recently, mNT and NAF-1 which are known to maintain
the levels of labile Fe and ROS were shown to be cooperating to control this homeostasis in mitochondria, and
this result confirms the presence of a direct link between
them. It may be that mNT transfers its cluster to NAF-1
and that this interaction regulates cellular proliferation and
apoptosis/autophagy activation [59].
Concluding remarks
This minireview focuses on the newly discovered [2Fe-2S]
protein family, the NEET proteins, which are involved in
numerous human pathologies and key cellular processes. We
described in detail the unique fold and structural elements of
these proteins and their uniquely labile [2Fe-2S] cluster that
play a key role in their function. Although in the last decade
a vast amount of information has been gathered about the
NEET proteins, key questions related to the NEET proteins
remain unsolved and await future studies. In particular, questions remain regarding structure–function, cluster lability,
protein partner interactions and drugs binding. We strongly
believe that coupling the experimental studies with computational simulations will pave the way toward answers to these
questions and to the comprehensive characterization of this
important NEET protein family.
Acknowledgements RN, RM, PAJ, JNO and PC acknowledge the support of NSF-MCB-1613462 (RM), BSF Grant 2015831 (RN), NIH
Grant GM101467 (PAJ), NSF-PHY-1427654 and NSF-CHE-1614101
(JNO), and of the Human Brain Project (PC).
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativeco
mmons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate
credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made.
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Affiliations
Ola Karmi1 · Henri‑Baptiste Marjault1 · Luca Pesce2,3 · Paolo Carloni2,3 · Jose’ N. Onuchic4,5 · Patricia A. Jennings6 ·
Ron Mittler7 · Rachel Nechushtai1
1
The Alexander Silberman Life Science Institute
and the Wolfson Center for Applied Structural Biology, The
Hebrew University of Jerusalem, Edmond J. Safra Campus
at Givat Ram, 91904‑0375 Jerusalem, Israel
2
Computational Biomedicine Section, Institute of Advanced
Simulation (IAS‑5) and Institute of Neuroscience
and Medicine (INM‑9), Forschungszentrum Jülich GmbH,
52425 Jülich, Germany
3
Department of Physics, RWTH-Aachen University,
52056 Aachen, Germany
4
Center for Theoretical Biological Physics, Rice University,
Houston, TX 77005, USA
13
5
Departments of Physics and Astronomy, Chemistry
and Biosciences, Rice University, Houston, TX 77005, USA
6
Department of Chemistry and Biochemistry, University
of California San Diego, La Jolla, CA 92093, USA
7
Department of Biological Sciences and BioDiscovery
Institute, University of North Texas, Denton, TX 76203, USA