Microbial "mitoNEET" homologs
The mammalian mitochondrial outer membrane protein, named "mitoNEET" [on the basis of its subcellular location on the outer membrane of mitochondria and the internal amino-acid sequence stretch of the mouse and human proteins, Asn-Glu-Glu-Thr (NEET)], was identified as a possible target protein of pioglitazone by cross-linking with a photoaffinity probe by Colca et al. The drug pioglitazone is a member of the thiazolidinedione class of insulin sensitizers for the treatment of type II diabetes, a complex metabolic disease of prevailing public health concern that is characterized in the initial stage by insulin resistance. Deficiency of mitoNEET expression in mice results in a compromise in the respiratory capacity of cardiac mitochondria.
Recent crystallographic studies on the recombinant soluble (CDGSH-type zinc-finger-like) domain of human mitoNEET at 1.4-1.8 Å resolution (PDB codes: 2qd0, 2r13, 2qh7 and 3ew0) have revealed that the recombinant mitoNEET soluble domain is a homodimer with each subunit binding a unique [2Fe–2S] cluster (but no Zn2+) in a three-cysteine plus one-histidine ligand environment. A very similar mammalian protein called Miner1/ERIS/CDGSH2, originally annotated as a CDGSH-type zinc-finger protein of unknown function, is also a member of the mitoNEET-like [2Fe-2S](Cys)3(His)1 protein superfamily, and localizes to both the outer membrane of mitochondria and the endoplasmic reticulum (with a 6:1 ratio). Mis-splicing of the mRNA that encodes this Miner1/ERIS protein is causative in the genetic disease Wolfram Syndrome 2 (WFS2). Knock-out mice are also symptomatic, with similar signs of early aging and optical atrophy.
Despite the potential importance in pharmacology and clinical medicine, the in vivo function of the mitoNEET protein superfamily remains elusive. We are interested in the structure and (redox) function of this unique iron-sulfur protein superfamily.
Biophysical studies on rat mitoNEET: spin-spin interactions between two [2Fe-2S] clusters
Interest in the redox chemistry and physiology of the mitoNEET superfamily has been heightened by the recent availability of crystal structures (Fig. 1). However, information about the electronic structure and interaction of this novel "twin" [2Fe-2S] cluster system was very limited, and the site of iron reduction and the cluster's magnetic interaction with the protein environment which account for the electron transfer function and mechanism remained elusive in the mammalian mitoNEET system.
Figure 1. (a) Relative location of two clusters in the human mitoNEET (soluble domain) dimeric structure (2QH7.pdb). In this structure, the outermost iron is coordinated by solvent-exposed His87 and Cys83 ligands, and the innermostiron is coordinated by Cys74 and Cys72 ligands. (b) X-band continuous-wave (CW) electron paramagnetic resonance (EPR) spectrum of reduced rat mitoNEET soluble domain at 20 K. Additional weak wings, probably resulting from magnetic interaction of the reduced clusters, accompany the main EPR signal on the high and low field sides. Microwave frequency and power, 9.037 GHz and 0.01 mW; sample concentration, ~4 mM (per protomer).
Strong antiferromagnetic coupling between the electron spins of two irons via a bridging structure produces an EPR-silent (S=0) ground state in the oxidized Fe3+-Fe3+ form of the [2Fe-2S] cluster and a paramagnetic S=1/2 ground state in the reduced Fe3+-Fe2+ form. Thus, the reduced [2Fe-2S] cluster in frozen solution is characterized by the anisotropic EPR spectrum, typically as a result of a rhombic g-tensor. The distinguishing feature between the reduced plant ferredoxin [2Fe-2S](Cys)4 and Rieske [2Fe-2S](Cys)2(His)2 clusters is the average value of the g-tensor components gav, which is ~1.96 for plant ferredoxins and ~1.91 for Rieske proteins. The cluster in the mitoNEET protein family is the first example of a "redox-active" [2Fe-2S] cluster with asymmetrical coordination of one of the irons (Fig. 1). Before the discovery of the mitoNEET protein family, we had rationally engineered a [2Fe-2S](Cys)3(His)1 environment into the hyperthermophilic archaeal Rieske-type ferredoxin (ARF) scaffold of Sulfolobus solfataricus (DDBJ-EMBL-GenBank code, AB047031) with replacement of the His64→Cys ligand (H64C-ARF). This accommodated a fairly thermostable [2Fe-2S] cluster in the oxidized form. However, its high sensitivity to dithionite reduction even under anaerobic conditions (leading to irreversible reductive cluster breakdown) precluded detailed characterization of its reduced state. Thus, the mammalian mitoNEET system opens a new path to explore a deeper analysis of the paramagnetic form of a unique (and redox-active) [2Fe-2S](Cys)3(His)1 cluster.
Continuous-wave (CW) X-band EPR spectrum of the dithionite-reduced, the rat mitoNEET soluble domain (residues 32-108; NCBI_GeneID code 294362), containing two nominally identical [2Fe-2S](Cys)3(His)1 clusters per dimer, possesses an anisotropic lineshape with a width of ~250 G, corresponding to rhombic g-tensor (Fig. 1b). The principal values of the g-tensor can be estimated by the values gz=2.005, gy=1.937, and gx=1.895. The average value of these components (gav ~1.945) is reasonably between the values of the plant ferredoxin (~1.96) and Rieske (~1.91) [2Fe-2S] clusters. In addition, EPR spectrum shows the splitting ~19 G of the gz component in the low field of the spectrum and a broadening of the central gy component from the poorly resolved splitting of the order ~10 G (Fig. 1b). These features resulted from the interaction of the electron spins of two adjacent, reduced clusters in the dimeric unit (Fig. 1). This interaction was not recognized in the previously published spectrum of the reduced mitoNEET soluble domain.
X-ray crystal structure of the human mitoNEET soluble domain gives distances of 14.3 Å and 17.7 Å between the innermost and outermost iron pairs of two [2Fe-2S] clusters, respectively (Fig. 1a). The distance between the geometrical centers of the two clusters is 15.9 Å. The parameter D=g^2β^2/r^3 =18500/r^3 (D in Gauss, r in Å), which characterizes an order of spin-spin interaction between two reduced clusters in the approximation considering them as two S=1/2 dipoles, varies within the interval 7.7 and 3.3 G for these distances (4.6 G for the center). Thus, the observed large gz splitting ~19 G (Fig. 1b) cannot be explained by the simple dipole-dipole interaction of two S=1/2 spins separated by the reported crystallographic distance (~14-18 Å) of the two adjacent clusters (Fig. 1a).
Our calculations demonstrate that the observed gz splitting in the EPR spectrum of the reduced rat mitoNEET is more adequately described by employing the “local spin model” which considers the spin-spin interactions between all four irons in reduced clusters (Fig. 2a). This model favors the reduction of the outermost iron with His87 and Cys83 ligands (Fig. 2b). These peculiarities suggest that the mammalian mitoNEET might have evolved to conduct rapid intramolecular electron transfer reaction in the dimeric unit.
Figure 2. Schematic presentation of vectors and angles for the qualitative calculation by employing the local spin model (a). In this figure, the r22 and r33 form the same angle θ with the external magnetic field vector Ho. ϕ is the angle between the planes containing the vectors (r23,r22) and (r22,Ho) and is equal to that between their normals n (to the plane containing r23 and r22) and m (to the plane containing r22 and Ho). The deviation of m from n depends on the orientation of Ho. In panels (b) and (c) prepared in 3D presentation by Prof. Sergei Dikanov (University of Illinois at Urbana-Champaign), the effective distance reff (Å) was calculated as a function of angles θ and ϕ (with the step 1°) when the outermost iron pair (reff ~11 Å for most orientations of the magnetic field relative to the molecular axes of the cluster defined by angles θ and ϕ) (b) or the innermost iron pair (reff ~22 Å, which is substantially longer) (c) undergoes the reduction in rat mitoNEET. Further details are given in the original article of this work.
2D pulsed EPR (HYSCORE) characterization of rat mitoNEET
Reduction of the outermost iron pair with His87 and Cys83 ligands in the mixed-valent state of the mitoNEET [2Fe-2S] cluster, indicated by local spin model analysis (Fig. 2), can be addressed independently by the two-dimensional pulsed EPR (also called hyperfine sublevel correlation, HYSCORE) characterization of hyperfine couplings from directly coordinated nitrogen of the His87 ligand. The representative 14N HYSCORE spectrum of the reduced 14N(N/A)-rat mitoNEET is shown in Fig. 3a. It consists of two quadrants containing cross-peaks from several nitrogens. Previous analysis with different values of 14N hyperfine couplings allows us to assign extended cross-peaks in the (+–) quadrant to strongly coupled (coordinated) 14Nδ of the histidine ligand (His87), and two pairs of cross-peaks in the (++) quadrant with an approximately circular shape of small radius to other, non-liganding nitrogens. The 14Nδ nuclear frequencies from the His87 ligand of rat mitoNEET, determined from the 14N HYSCORE spectra recorded at the fields corresponding to principal values of the g-tensor, provide an estimate of diagonal components of the hyperfine and quadrupole tensors in the g-tensor coordinates and the isotropic hyperfine constant (14)a=6.1 MHz.
Figure 3. HYSCORE spectra in contour presentation of the reduced [2Fe-2S] cluster in 14N(N/A, 99.63%) (a) and uniformly 15N-labeled (b) rat mitoNEET soluble domain; dd, ds, and ss are cross-peaks correlating d, double-quantum, and s, single-quantum transitions. Magnetic field, time τ, and microwave frequency were: 3672 G, 136 ns, 9.706 GHz (a); 3580 G, 136 ns, 9.704 GHz (b).
The 15N HYSCORE spectrum of the uniformly 15N-labeled rat mitoNEET (Fig. 3b) shows only one pair of cross-peaks in the (+−) quadrant from the His87 15Nδ (nuclear spin I=1/2, no quadrupole moment). The isotropic and anisotropic parts of axial hyperfine tensor (15)a=8 MHz, (15)T=1.1 MHz (corresponding to (14)a=5.7 MHz and (14)T=0.8 MHz) defining principal values of the tensor were obtained by the contour lineshape analysis (data not shown). Notably, these parameters for His87 Nδ in rat mitoNEET are only ~10% above the corresponding values for one of the two Nδ(His) 's (with larger hyperfine tensor) in Rieske-type proteins (see also the original article of this work). In the Rieske-type protein system, the Nδ(His) hyperfine tensors depend on anisotropic interaction with the electron spins of the Fe(II) and Fe(III), and the spin density transferred onto the strongly coupled (i.e., coordinated) 14Nδ(His) atom. The constrains on Nδ(His) location (stable Fe-Nδ(His) distances and angles between Fe-Fe and Fe-Nδ(His)) based on the available crystal structures would account for the similarity of hyperfine tensors between different proteins, if they are determined mainly by the location of the Nδ(His) nuclei relative to the electron spins of the cluster, and are relatively insensitive to variations in orientation of the imidazole plane. The latter influences only the overlap of the iron and nitrogen orbitals responsible for the spin density transfer onto the ligand. This means that the hyperfine tensor of the strongly coupled Nδ(His) and its principal directions mainly reflect the position of the Nδ(His) atom itself relative to the iron-sulfur cluster, and therefore would not provide direct information about changes in orientation of the imidazole plane, so long as the Nδ(His) location remains approximately constant. Thus, the HYSCORE analysis provides the second indication that the Fe2+ site in the reduced cluster of rat mitoNEET is the outermost iron.
It should be added that maximum hyperfine and quadrupolar splittings from 14Nδ(His87) in rat mitoNEET are observed along gx(min) axis, in contrast to the gz(max) axis for both 14Nδ(His) ligands in Rieske-type proteins. The hyperfine tensor of the directly coordinated Nδ(His) and its principal directions mainly reflect the position of the nitrogen atom itself relative to the iron-sulfur cluster, whereas the principal directions of the nuclear quadrupole interaction tensor are determined by the geometry of the electron orbitals around the nucleus and are associated with the ligand molecule itself. Both mitoNEET- and Rieske-type cluster systems have one common Nδ(His) ligand located at the equivalent position relative to the [2Fe-2S] cluster (as judged by similar Fe-Nδ(His) distances, angles between the Fe-Fe and Fe-Nδ(His) and orientations of imidazole plane), which would account for the similar hyperfine and quadrupole tensors (see the original article of this work). Based on these considerations, it seems reasonable to postulate a possible difference in orientation of the gmax, gmid, and gmin principal axes of the g-tensors relative to the basic molecular axes between the mitoNEET and Rieske cluster systems.
14N and 15N HYSCORE characterization of rat mitoNEET also suggested marked differences between the mitoNEET and Rieske [2Fe-2S] protein systems in regard to the cluster's magnetic interaction with the protein environment (Figs. 4-6). In contrast to Rieske proteins, we did not detect (non-coordinated) peptide nitrogen (Np(s)) with hyperfine couplings significantly exceeding the Nε(His) coupling. It is likely that peptide nitrogens involved in hydrogen-bond formation with the reduced cluster of the mammalian mitoNEET soluble domain carry less spin density than those of Rieske-type proteins, reflecting their structural differences, including configurations of the liganding residues to the reduced cluster. In conjunction with the marked instability of a reduced [2Fe-2S](Cys)3(His)1 cluster rationally engineered into the archaeal Rieske-type ferredoxin scaffold, our results may imply the importance of the cluster's interaction with the protein environment for the assurance of biological redox function. Although the extensive nuclear magnetic resonance (NMR) studies on bacterial rubredoxin have revealed some quantitative relations involving unpaired spin density onto peptide nitrogens, strength of hydrogen bonds, and redox properties, NMR identification and full assignment of resonances from a paramagnetic center of a polynuclear iron-sulfur protein with slow electronic relaxation rates (e.g. the [2Fe-2S](Cys)4 ferredoxins) are still challenging. Because up to now the parametrization of weakly coupled subsystems has not yet been optimized at the density functional theory level, the present results can also be used in theoretical analysis for selection of an appropriate model of the mixed-valence state of the mammalian mitoNEET system.
Figure 4. The (++) quadrant of the 14N HYSCORE spectrum taken from Fig. 3a (a); 3D stacked presentation of the line from weakly coupled 15N nuclei in the (++) quadrant of 15N HYSCORE spectrum (recorded near gy) from Fig. 3b (b); the same as (b) for uniformly 15N-labeled, reduced rat mitoNEET (c) and a low-potential, archaeal Rieske-type ferredoxin (ARF) from S. solfataricus (d). Both (c) and (d) were recorded near gz area of the EPR line. Magnetic field, time τ, and microwave frequency, respectively: 3450 G, 136 ns, 9.704 GHz (c); 3425 G, 136 ns, 9.690 GHz (d). Notably, the intensity of the central “matrix” peak at the diagonal point (15νN,15νN) is suppressed in (c) and (d) in contrast with (b).
Figure 5. 3D-stacked presentation of the HYSCORE spectra in the (++) quadrant of 14N(N/A)-rat mitoNEET (a) and partially and selectively 15N(3)-His-labeled rat mitoNEET (b). The two-pulse ESEEM amplitude of the 15N(3)-His-labeled rec mitoNEET (b) showed ~30% substitution of the coordinated His87 14Nδ by 15N (not shown). In the (++) quadrant of the HYSCORE spectra, this sample shows a new doublet (15N) located symmetrically around diagonal point (15νN,15νN) with the splitting ~0.45-0.55 MHz [and varying width up to 0.2 MHz due to difference in contributing orientations] (b). The appearance of this doublet is accompanied by ~30% decrease of the relative intensity of the cross-peaks 2 relative to the cross-peaks 1 (indicated by arrows in (b)). Spectra were recorded at similar conditions with magnetic field 3620 G, time τ=136 ns, microwave frequency ~9.70 GHz.
Figure 6. 3D presentation of the HYSCORE spectra in the (++) quadrant of the partially and selectively 15N(3)-His-labeled rat mitoNEET soluble domain recorded near gx (a) and gy (c), and superimposition of the 3D plots for the uniformly 15N-labeled rat mitoNEET (blue) and the difference [uniformly 15N-labeled minus 15N(3)-His-labeled rat mitoNEET multiplied by coefficient 3.5 (i.e., corrected for ~30% substitution efficiency for 15N(3)-His), see Fig. 5] spectra (red) near gx (b) and gy (d). The difference spectra (b,d) indicate ~25% contribution of His87 15Nε to the amplitude of major doublet with the splitting ~0.4-0.5 MHz in gx (b) and gz (not shown) area and less than ~10% contribution in gy (d) area. This experiment also indicates that scrambling of randomly 15N-labeled peptide and other side chain nitrogens around the cluster does not occur with this partially 15N(3)-His-labeled sample (a,c), which would contribute to the central diagonal point (15νN,15νN) and to areas around this peak in the HYSCORE spectra [as seen with the uniformly 15N-labeled protein (b,d)]. Spectra were recorded at similar conditions with magnetic field 3670 G (near gx) for (a,b), and 3580 G (near gy) for (c,d); time τ=136 ns; microwave frequency ~9.70 GHz.
Crystallization of TthNEET0026
Thermus thermophilus strain HB8 (JCM10941) is a Gram-negative thermophilic bacterium isolated from the Mine hot springs, Izu, Japan. The whole genomic DNA sequence has been analyzed (http://www.thermus.org) and genetic manipulation systems for the introduction, disruption and homologous expression of intrinsic/foreign genes have been developed. A homology search of the deduced human and rat mitoNEET sequences against the complete T. thermophilus HB8 genome database using the NCBI BLAST and Kyoto Encyclopedia of Genes and Genomes (KEGG) servers indicated at least two hypothetical genes, ttha0026 and ttha1309, that code for mitoNEET-like soluble proteins (our unpublished results). The ttha1309 gene is tentatively annotated as a short antiframe (ORF) located within a hypothetical gene cluster coding for a putative radical SAM enzyme family (data not shown). The ttha0026 gene is a part of the (hypothetical) ttha0025–ttha0026 cluster coding for a putative leucine-rich membrane protein (TTHA0025; 157 amino acids) and a mitoNEET-like water-soluble protein (TTHA0026; 69 amino acids) (Fig. 7). Moreover, the four ligand residues (Cys37, Cys39, Cys48 and His52) in the [2Fe–2S] cluster-binding motif of human mitoNEET are strictly conserved in the TTHA0026 protein (Fig. 7), even though TTHA0026 is tentatively categorized as a hypothetical CDGSH-type zinc-finger protein. It is therefore of particular interest to investigate whether TTHA0026 is indeed a bacterial prototypal model of mitoNEET carrying a mitoNEET-like [2Fe–2S] cluster and to address its functionalities through elucidation of its structure at atomic resolution and analyses of the phenotypes and the in vivo network of genetically manipulated T. thermophilus strains.
Figure 7. The Schematic organization of the ttha0025–ttha0026 gene cluster and its adjacent regions in the genomic DNA sequence of T. thermophilus HB8. There is no direct information available concerning the specificfunction of the products of the ttha0024–ttha0027 genes [TTHA0025 is a putative (leucine-rich) membrane protein, TTHA0026 is a water-soluble homologue of the mammalian mitoNEET soluble domain (TthNEET0026) and TTHA0027 is a putative potassium channel β-subunit homologue]. Multiple sequence alignment of selected mitoNEET-like proteins indicates the conservation of one histidine (indicated by a red arrow) and three cysteine residues, which provide the [2Fe–2S](Cys)3(His)1 cluster-binding motif (boxed) in the recombinant human mitoNEET soluble-domain fragment. The overall sequence identity between TthNEET0026 (69 amino acids) and the human mitoNEET soluble domain of known structure (76 amino acids) is 20.3%. Accession numbers: T. thermophilus HB8 TthNEET0026, ORFannotation/KEGG code TTHA0026, NCBI GeneID code 3168947; Rattus norvegicus (rat) mitoNEET (Cisd1), NCBI GeneID/KEGG code 294362; Homo sapiens (human) mitoNEET soluble-domain fragment, PDB codes 2qd0, 2r13 and 2qh7.
The thermophilic homolog of mitoNEET (TTHA0026) from T. thermophilus HB8 was heterologously overproduced in E. coli BL21-CodonPlus(DE3)-RIL strain and purified as a water-soluble prototypal protein containing the mitoNEET-like [2Fe-2S] cluster (Fig. 8). These results indicate the presence of the redox-active, mitoNEET-like dimeric [2Fe-2S](Cys)3(His)1 protein family across various organisms from thermophiles to mammals. The resultant recombinant protein, named TthNEET0026, has been crystallized in its oxidized form by the hanging-drop vapor diffusion method using 17%(w/v) polyethylene glycol 4000, 8.5%(v/v) 2-propanol, 15%(v/v) glycerol and 85 mM HEPES–NaOH buffer, pH 7.2 (Fig. 8). The dark reddish crystals diffract to 1.80 Å resolution and belong to the hexagonal space group P43212, with unit-cell parameters a = 45.51, c = 84.26 Å. The asymmetric unit contains one protein molecule.
Figure 8. (a) The visible–UVabsorption spectra of purified TthNEET0026 (red trace) and rat mitoNEET [2Fe–2S] cluster-binding domain (residues 32–108; black trace). The absorption maxima (nm) are indicated in the figure. (b) Typical crystals of recombinant TthNEET0026. The maximum dimensions of the crystals are approximately 0.1 X 0.1 X 0.15 mm.
The 1.80-Å structure of TthNEET0026