Crystallization and preliminary X-ray diffraction studies of the prototypal homologue of mitoNEET (<i>Tth</i>-NEET0026) from the extreme thermophile<i>Thermus thermophilus</i>HB8

Kounosu, Asako; Iwasaki, Toshio; Baba, Seiki; Hayashi-Iwasaki, Yoko; Oshima, Tairo; Kumasaka, Takashi

doi:10.1107/s1744309108035975

Cited by 10 publications

(13 citation statements)

References 15 publications

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“…The cells were pelleted by centrifugation, and the rec mitoNEET (residues 32-108) having a hexahistidine-tag plus a thrombin cleavage site was purified essentially as reported previously for archaeal Rieske [2Fe-2S] proteins and the Thermus thermophilus homolog of mammalian mitoNEET,7,9,10 except that the entire purification was performed at 4 °C using buffers adjusted at pH 8.0 and that the heat treatment of the crude cell lysate was omitted. The sample was further purified by gel-filtration chromatography (Sephadex G-75; Amersham Pharmacia Biotech) eluted at room temperature with 10 mM HEPES-NaOH buffer, pH 8.0, containing 500 mM NaCl, and then concentrated with Centriprep-10 and Microcon-YM10 apparatus (Amicon) to ~4 mM (per protomer), rapidly frozen in liquid nitrogen, and stored at −80 °C until use.…”

Section: Methodsmentioning

confidence: 99%

“…4–6 Although the integrity of a [2Fe-2S] cluster and the functional role of the His87 ligand in native mitoNEET in vivo remains unknown, its thermophilic bacterial water-soluble homolog from Thermus thermophilus HB8 has been overproduced in Escherichia coli , crystallized, and found to contain two adjacent, mitoNEET-like [2Fe-2S](Cys) 3 (His) 1 clusters per dimeric unit 7. 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 7. Interest in the redox chemistry and physiology of the mitoNEET superfamily has been heightened by the recent availability of crystal structures4–6 (Figure 1a).…”

Section: Introductionmentioning

confidence: 99%

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Continuous-Wave and Pulsed EPR Characterization of the [2Fe−2S](Cys)₃(His)₁ Cluster in Rat MitoNEET

et al. 2009

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CW EPR spectra of reduced [2Fe-2S](Cys)3(His)1 clusters of mammalian mitoNEET soluble domain appear to produce features resulting from the interaction of the electron spins of the two adjacent clusters, which can be explained by employing the local spin model. This model favors the reduction of the outermost iron with His87 and Cys83 ligands, which is supported by orientation-selected hyperfine sublevel correlation (HYSCORE) characterization of the uniformly 15N-labeled mitoNEET showing one strongly coupled nitrogen from the His87 Nδ ligand with hyperfine coupling 15a=8 MHz. The 14N and 15N HYSCORE spectra also exhibit at least two different cross-peaks located near diagonal in the (++) quadrant, with frequencies ~2.8 and 2.4 MHz (N2), and the other ~4.0 and 3.5 MHz (N1), but did not show any of the larger splitting ~1.1–1.4 MHz previously seen with Rieske proteins. Further analysis with partially 15N(3)-His-labeled protein indicates that His87 Nε cross-peaks produce resolved features (N2) in the 14N spectrum but contribute much less than weakly-coupled peptide nitrogen species to the (++) quadrant in the 15N spectrum. It is suggested that these quantitative data may be used in future functional and theoretical studies on the mammalian mitoNEET [2Fe-2S] cluster system.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Continuous-Wave and Pulsed EPR Characterization of the [2Fe−2S](Cys)₃(His)₁ Cluster in Rat MitoNEET

et al. 2009

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“…1 Fe/S proteins involved in electron-transfer processes can be classified on basis of their structural and electrochemical properties. 2 A prominent class of such proteins are the [2Fe-2S] ferredoxins, containing two iron ions that are bridged by two inorganic sulfides and usually coordinated by four cysteine thiolates. [2Fe-2S] ferredoxins share a -grasp structure composed of 3-5 -strands with 1-3 -helices.…”

Section: Introductionmentioning

confidence: 99%

Apd1 and Aim32 Are Prototypes of Bishistidinyl-Coordinated Non-Rieske [2Fe–2S] Proteins

et al. 2019

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Apd1, a cytosolic yeast protein, and Aim32, its counterpart in the mitochondrial matrix, have a C-terminal thioredoxinlike ferredoxin domain and a widely divergent N-terminal domain. These proteins are found in bacteria, plants, fungi and unicellular pathogenic eukaryotes, but not in Metazoa. Our chemogenetic experiments demonstrate that the highly conserved cysteine and histidine residues within the C-X8-C-X24-75-H-X-G-G-H motif of the TLF domain of Apd1 and Aim32 proteins are essential for viability upon treatment of yeast cells with the redox potentiators gallobenzophenone or pyrogallol, respectively. UV-Vis, EPR and Mössbauer spectroscopy of purified wild type Apd1 and three His to Cys variants demonstrated that Cys207 and Cys216 are the ligands of the ferric ion and His255 and His259 are the ligands of the reducible iron ion of the [2Fe-2S] 2+/1+ cluster. The [2Fe-2S] center of Apd1 (Em,7 = 164±5 mV, pKox1,27.9±0.1 and 9.7±0.1) differs from both dioxygenase (Em,7  150 mV, pKox1,2=9.8 and 11.5) and cytochrome bc1/b6f Rieske clusters (Em,7  +300 mV, pKox1,2= 7.7 and 9.8). Apd1 and its engineered variants represent an unprecedented flexible system for which a stable [2Fe-2S] cluster with two histidine ligands, (two different) single histidine ligands or only cysteinyl ligands is possible in the same protein fold. Our results define a remarkable example of convergent evolution of [2Fe-2S] cluster containing proteins with bis-histidinyl coordination and proton-coupled electron transfer. Strain Plasmid Gallobenzophenone 25 µM none 50 µM empty Aim32 ∆aim32 Wild type Apd1 empty ∆apd1 empty Aim32 Apd1 a empty ∆sod2 Strain Plasmid ∆sod2/ Δaim32 empty Aim32 Apd1 Pyrogallol 25 µM none 50 µM empty Aim32 ∆aim32 Wild type

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“…To date, structures are available only for the type 1 CISD proteins mitoNEET and Miner1. Preliminary crystallization results, but no structure, have been reported for a type 4 CISD [19]. To investigate the structure of different types of CISDs, we have determined high-resolution crystal structures for types 3, 4, and 6 CISDs and predicted the structures for type 2 and 7 CISDs.…”

Section: Introductionmentioning

confidence: 99%

Structure and Molecular Evolution of CDGSH Iron-Sulfur Domains

et al. 2011

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The recently discovered CDGSH iron-sulfur domains (CISDs) are classified into seven major types with a wide distribution throughout the three domains of life. The type 1 protein mitoNEET has been shown to fold into a dimer with the signature CDGSH motif binding to a [2Fe-2S] cluster. However, the structures of all other types of CISDs were unknown. Here we report the crystal structures of type 3, 4, and 6 CISDs determined at 1.5 Å, 1.8 Å and 1.15 Å resolution, respectively. The type 3 and 4 CISD each contain one CDGSH motif and adopt a dimeric structure. Although similar to each other, the two structures have permutated topologies, and both are distinct from the type 1 structure. The type 6 CISD contains tandem CDGSH motifs and adopts a monomeric structure with an internal pseudo dyad symmetry. All currently known CISD structures share dual iron-sulfur binding modules and a β-sandwich for either intermolecular or intramolecular dimerization. The iron-sulfur binding module, the β-strand N-terminal to the module and a proline motif are conserved among different type structures, but the dimerization module and the interface and orientation between the two iron-sulfur binding modules are divergent. Sequence analysis further shows resemblance between CISD types 4 and 7 and between 1 and 2. Our findings suggest that all CISDs share common ancestry and diverged into three primary folds with a characteristic phylogenetic distribution: a eukaryote-specific fold adopted by types 1 and 2 proteins, a prokaryote-specific fold adopted by types 3, 4 and 7 proteins, and a tandem-motif fold adopted by types 5 and 6 proteins. Our comprehensive structural, sequential and phylogenetic analysis provides significant insight into the assembly principles and evolutionary relationship of CISDs.

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Crystallization and preliminary X-ray diffraction studies of the prototypal homologue of mitoNEET (Tth-NEET0026) from the extreme thermophileThermus thermophilusHB8

Cited by 10 publications

References 15 publications

Continuous-Wave and Pulsed EPR Characterization of the [2Fe−2S](Cys)₃(His)₁ Cluster in Rat MitoNEET

Continuous-Wave and Pulsed EPR Characterization of the [2Fe−2S](Cys)₃(His)₁ Cluster in Rat MitoNEET

Apd1 and Aim32 Are Prototypes of Bishistidinyl-Coordinated Non-Rieske [2Fe–2S] Proteins

Structure and Molecular Evolution of CDGSH Iron-Sulfur Domains

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Crystallization and preliminary X-ray diffraction studies of the prototypal homologue of mitoNEET (Tth-NEET0026) from the extreme thermophileThermus thermophilusHB8

Cited by 10 publications

References 15 publications

Continuous-Wave and Pulsed EPR Characterization of the [2Fe−2S](Cys)3(His)1 Cluster in Rat MitoNEET

Continuous-Wave and Pulsed EPR Characterization of the [2Fe−2S](Cys)3(His)1 Cluster in Rat MitoNEET

Apd1 and Aim32 Are Prototypes of Bishistidinyl-Coordinated Non-Rieske [2Fe–2S] Proteins

Structure and Molecular Evolution of CDGSH Iron-Sulfur Domains

Contact Info

Product

Resources

About

Continuous-Wave and Pulsed EPR Characterization of the [2Fe−2S](Cys)₃(His)₁ Cluster in Rat MitoNEET

Continuous-Wave and Pulsed EPR Characterization of the [2Fe−2S](Cys)₃(His)₁ Cluster in Rat MitoNEET