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The iron–sulfur cluster assembly (ISC) protein Iba57 executes a tetrahydrofolate-independent function in mitochondrial [4Fe–4S] protein maturation

Open AccessPublished:September 05, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102465
      Mitochondria harbor the bacteria-inherited iron–sulfur cluster assembly (ISC) machinery to generate [2Fe–2S; iron–sulfur (Fe–S)] and [4Fe–4S] proteins. In yeast, assembly of [4Fe–4S] proteins specifically involves the ISC proteins Isa1, Isa2, Iba57, Bol3, and Nfu1. Functional defects in their human equivalents cause the multiple mitochondrial dysfunction syndromes, severe disorders with a broad clinical spectrum. The bacterial Iba57 ancestor YgfZ was described to require tetrahydrofolate (THF) for its function in the maturation of selected [4Fe–4S] proteins. Both YgfZ and Iba57 are structurally related to an enzyme family catalyzing THF-dependent one-carbon transfer reactions including GcvT of the glycine cleavage system. On this basis, a universally conserved folate requirement in ISC-dependent [4Fe–4S] protein biogenesis was proposed. To test this idea for mitochondrial Iba57, we performed genetic and biochemical studies in Saccharomyces cerevisiae, and we solved the crystal structure of Iba57 from the thermophilic fungus Chaetomium thermophilum. We provide three lines of evidence for the THF independence of the Iba57-catalyzed [4Fe–4S] protein assembly pathway. First, yeast mutants lacking folate show no defect in mitochondrial [4Fe–4S] protein maturation. Second, the 3D structure of Iba57 lacks many of the side-chain contacts to THF as defined in GcvT, and the THF-binding pocket is constricted. Third, mutations in conserved Iba57 residues that are essential for THF-dependent catalysis in GcvT do not impair Iba57 function in vivo, in contrast to an exchange of the invariant, surface-exposed cysteine residue. We conclude that mitochondrial Iba57, despite structural similarities to both YgfZ and THF-binding proteins, does not utilize folate for its function.

      Keywords

      Abbreviations:

      CtIba57 (Chaetomium thermophilum Iba57), dTMP (deoxythymidine monophosphate), EcYgfZ (Escherichia coli YgfZ), Fe-S (iron–sulfur), HsGcvT (human GcvT), HsIBA57 (human IBA57), ISC (iron–sulfur cluster assembly), PDB (protein data bank), THF (tetrahydrofolate)
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      Results

      Folate is not required for Fe–S protein maturation in yeast

      Based on studies with EcYgfZ, a universal role for THFs in the formation of iron-sulfur clusters was postulated (
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      ). In Saccharomyces cerevisiae, mutants with defects in folate biosynthesis are viable when supplemented with adenine, His, Met, and deoxythymidine monophosphate (dTMP) (
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      ). However, of these four folate-requiring supplements, only the biosynthesis of methionine involves iron-sulfur cluster-dependent enzymes, yet the specific folate-requiring step does not involve Fe–S proteins (
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      Toward a better understanding of folate metabolism in health and disease.
      ). In contrast, deletion of the ygfZ homolog IBA57 in S. cerevisiae is associated with auxotrophies for Glu and Lys that are caused by loss of function of the mitochondrial [4Fe–4S] proteins aconitase and homoaconitase, respectively (
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      Mitochondrial Iba57p is required for Fe/S cluster formation on aconitase and activation of radical SAM enzymes.
      ). These auxotrophies were not reported previously for any folate biosynthesis mutant in S. cerevisiae, which puts the postulated role of THF in the enzymatic activity of mitochondrial Iba57 into question (
      • Bayly A.M.
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      • Hankins E.G.
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      ,
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      Mitochondrial one-carbon metabolism is adapted to the specific needs of yeast, plants and mammals.
      ,
      • Locasale J.W.
      Serine, glycine and one-carbon units: Cancer metabolism in full circle.
      ).
      In order to resolve this point, we analyzed yeast strains with defects in THF biosynthesis and/or utilization for potential defects in mitochondrial Fe–S protein maturation. First, we chose four mutants with gene deletions of key components of mitochondrial THF metabolism: Met13, the mitochondrial methylenetetrahydrofolate reductase; Met7, the folylpolyglutamate synthetase required for methionine synthesis and mitochondrial DNA maintenance (
      • Cherest H.
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      Polyglutamylation of folate coenzymes is necessary for methionine biosynthesis and maintenance of intact mitochondrial genome in Saccharomyces cerevisiae.
      ); Mis1, the mitochondrial C1-THF synthase; and Ade3, the cytosolic counterpart of Mis1. Furthermore, we created an ade3Δ/mis1Δ double mutant that is unable to produce 5,10-methenyltetrahydrofolate (
      • Christensen K.E.
      • MacKenzie R.E.
      Mitochondrial one-carbon metabolism is adapted to the specific needs of yeast, plants and mammals.
      ,
      • Locasale J.W.
      Serine, glycine and one-carbon units: Cancer metabolism in full circle.
      ). Yet, despite their strong defects in one-carbon metabolism, all these mutants were perfectly able to grow on minimal medium lacking Lys and Glu, clearly differing from an aconitase (Aco1) deletion mutant (Fig. 1A). Next, we analyzed yeast mutants deleted for FOL2 encoding the GTP-cyclohydrolase I that catalyzes the first step in folate biosynthesis (
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      • et al.
      GTP cyclohydrolase I and tyrosine hydroxylase gene mutations in familial and sporadic dopa-responsive dystonia patients.
      ). We created two fol2Δ strains by independent deletion strategies in order to ensure that the observed phenotypes were not because of secondary effects caused by this massive impact in metabolism. Both mutants were unable to grow in rich or minimal medium lacking folate, unless supplemented with the four folate-requiring supplements, in particular dTMP (Fig. 1B and Fig. S1A). Furthermore, the fol2Δ strains were unable to grow on the nonfermentable carbon source glycerol (Fig. S1B). Mating experiments with a rho0 tester strain indicated the loss of mitochondrial DNA as a result of the FOL2 deletion. However, unlike iba57Δ cells that are also rho0, the fol2Δ strains did not require Lys or Glu for growth (Fig. 1B) (
      • Gelling C.
      • Dawes I.W.
      • Richhardt N.
      • Lill R.
      • Muhlenhoff U.
      Mitochondrial Iba57p is required for Fe/S cluster formation on aconitase and activation of radical SAM enzymes.
      ). The slower growth of the fol2Δ mutants under this condition was likely because of their rho0 phenotype (cf. Fig. S1B). Consistently, fol2Δ cells displayed wildtype aconitase activities (Fig. 1C), and the lipoylation of mitochondrial pyruvate- and α-ketoglutarate-dehydrogenase subunits Lat1 and Kgd2, respectively, was normal (Fig. 1D). Both aconitase and the radical-SAM enzyme lipoyl synthase (Lip5) are mitochondrial [4Fe–4S] proteins and essentially require Iba57 for maturation (
      • Gelling C.
      • Dawes I.W.
      • Richhardt N.
      • Lill R.
      • Muhlenhoff U.
      Mitochondrial Iba57p is required for Fe/S cluster formation on aconitase and activation of radical SAM enzymes.
      ,
      • Sheftel A.D.
      • Wilbrecht C.
      • Stehling O.
      • Niggemeyer B.
      • Elsasser H.P.
      • Muhlenhoff U.
      • et al.
      The human mitochondrial ISCA1, ISCA2, and IBA57 proteins are required for [4Fe-4S] protein maturation.
      ). Taken together, these data show that folate, in contrast to Iba57, is not required for the biogenesis of mitochondrial [4Fe–4S] proteins.
      Figure thumbnail gr1
      Figure 1Folate is not required for mitochondrial [4Fe–4S] protein biogenesis in Saccharomyces cerevisiae. A, the indicated yeast strains (BY4741 background; MATa his3Δ leu2Δ met15Δ ura3Δ) with defects in the utilization or modification of folate were cultivated on folate-free synthetic defined (SD) minimal medium containing the four folate-dependent supplements Ade, His, Met, and dTMP in the presence or the absence of Lys and Glu. BY4742 (MATα his3Δ leu2Δ lys2Δ ura3Δ) and aco1Δ (W303-1A background) requiring supplementation with either Lys or Glu served as control. B, two independent folate-auxotroph fol2Δ deletion strains (W303-1A background) were streaked onto folate-free SD minimal medium agar plates plus Ade, His, Met, and dTMP in the presence or the absence of Lys and Glu. C and D, cells cultivated in folate-free SD minimal medium plus Ade, His, Met, and dTMP were assayed for (C) aconitase enzyme activities (relative to malate dehydrogenase [MDH]) in cell extracts and (D) the lipoylation (LA) of the E2 subunits of pyruvate (Lat1) and 2-ketoglutarate dehydrogenases (Kgd2) by immunostaining of isolated mitochondrial extracts. An unspecific band crossreacting with the anti-LA antibody served as a loading control. W303-1A wildtype and rho0 cells as well as iba57Δ were used as controls. Error bars indicate the SD (n ≥ 4). dTMP, deoxythymidine monophosphate.
      In order to test whether the folate-dependent bacterial YgfZ can replace yeast Iba57 in mitochondrial Fe–S protein biogenesis, we expressed EcYgfZ with an N-terminal mitochondrial targeting sequence in iba57Δ cells. However, these transformed cells remained unable to grow on minimal medium lacking Lys and Glu, and aconitase activities were not restored, even when EcYgfZ was overproduced from a p424 high-copy vector (Fig. 2, A and B). This did not change when E. coli IscA, a likely partner of EcYgfZ, was coexpressed together with EcYgfZ. The localization of EcYgfZ in mitochondria of iba57Δ cells was verified by expressing EcYgfZ with a C-terminal Myc tag from low- (p416) or high-copy (p426) vectors (Fig. 2C). Like EcYgfZ, EcYgfZ-Myc also did not rescue the growth defect of iba57Δ cells (Fig. S2). Apparently, EcYgfZ cannot replace Iba57 in yeast, although vice versa yeast Iba57 can substitute EcYgfZ in E. coli, albeit poorly (
      • Waller J.C.
      • Alvarez S.
      • Naponelli V.
      • Lara-Nunez A.
      • Blaby I.K.
      • Da Silva V.
      • et al.
      A role for tetrahydrofolates in the metabolism of iron-sulfur clusters in all domains of life.
      ). This observation is reminiscent of complementation studies with the plastid version of Arabidopsis thaliana IBA57, which could functionally replace EcYgfZ in E. coli but not Iba57 in yeast (
      • Waller J.C.
      • Alvarez S.
      • Naponelli V.
      • Lara-Nunez A.
      • Blaby I.K.
      • Da Silva V.
      • et al.
      A role for tetrahydrofolates in the metabolism of iron-sulfur clusters in all domains of life.
      ,
      • Uzarska M.A.
      • Przybyla-Toscano J.
      • Spantgar F.
      • Zannini F.
      • Lill R.
      • Muhlenhoff U.
      • et al.
      Conserved functions of Arabidopsis mitochondrial late-acting maturation factors in the trafficking of iron sulfur clusters.
      ).
      Figure thumbnail gr2
      Figure 2Escherichia coli YgfZ does not complement Saccharomyces cerevisiae iba57Δ cells. A, strain iba57Δ (W303-1A background) was transformed with the indicated combinations of plasmids () that allow the expression of E. coli YgfZ and IscA in mitochondria under the control of the TDH3 promoter. Cells were cultivated on synthetic defined (SD) minimal medium agar plates in the presence or the absence of Lys and Glu. B, cells were cultivated in SD minimal medium, and cell extracts were assayed for aconitase activities. iba57Δ expressing yeast IBA57 from a plasmid and cells with empty vector served as controls. Error bars indicate the standard deviation (n ≥ 4). C, iba57Δ cells harboring low-copy (p416) or high-copy (p426) vectors for the expression of EcYgfZ with a C-terminal Myc tag under the control of the TDH3 promoter were fractionated into mitochondria (Mit) and postmitochondrial supernatant (PMS) fractions. iba57Δ cells with the empty vector (p416) served as a control. Fractions were analyzed for the presence of EcYgfZ by immunostaining with α-Myc antibodies. The asterisks (∗) likely indicate the noncleaved EcYgfZ-Myc precursor. Stains for mitochondrial Aco1 and cytosolic Dre2 serve to document the quality of the fractionation. An unspecific protein stained with Fast Green FCF served as a loading control. EcYgfZ, Escherichia coli YgfZ.

      The 3D structure of mitochondrial Iba57 is incompatible with high-affinity THF binding

      To obtain structural criteria for whether mitochondrial Iba57 might be a THF-dependent enzyme, we solved the crystal structure of Iba57 from the thermophilic fungus Chaetomium thermophilum (Ct). The mature form of CtIba57 (residues 61–476) was expressed in E. coli BL21 (DE3) and purified via an N-terminal His tag by nickel–nitrilotriacetic acid affinity and size-exclusion chromatography. The crystal structure of CtIba57 (Protein Data Bank [PDB] code: 7Z3H) was solved by molecular replacement using the structure of human Iba57 (HsIBA57) as a search model (Table S1) (
      • Gourdoupis S.
      • Nasta V.
      • Calderone V.
      • Ciofi-Baffoni S.
      • Banci L.
      IBA57 recruits ISCA2 to form a [2Fe-2S] cluster-mediated complex.
      ). Similar to EcYgfZ, CtIba57 crystallized as a disulfide-bridged homodimer (
      • Teplyakov A.
      • Obmolova G.
      • Sarikaya E.
      • Pullalarevu S.
      • Krajewski W.
      • Galkin A.
      • et al.
      Crystal structure of the YgfZ protein from Escherichia coli suggests a folate-dependent regulatory role in one-carbon metabolism.
      ) (Fig. 3A). Since the conserved Cys304 is essential for function in [4Fe–4S] cluster formation in vitro (
      • Weiler B.D.
      • Bruck M.C.
      • Kothe I.
      • Bill E.
      • Lill R.
      • Muhlenhoff U.
      Mitochondrial [4Fe-4S] protein assembly involves reductive [2Fe-2S] cluster fusion on ISCA1-ISCA2 by electron flow from ferredoxin FDX2.
      ) (see also later), and since the interface area is rather small, the dimerization likely is a consequence of the long incubation time during crystallization. Each CtIba57 monomer consists of 9 α-helices and 16 β-strands forming three domains (
      • Zanello P.
      Structure and electrochemistry of proteins harboring iron-sulfur clusters of different nuclearities. Part IV. Canonical, non-canonical and hybrid iron-sulfur proteins.
      ,
      • Braymer J.J.
      • Freibert S.A.
      • Rakwalska-Bange M.
      • Lill R.
      Mechanistic concepts of iron-sulfur protein biogenesis in biology.
      ,
      • Lill R.
      • Freibert S.A.
      Mechanisms of mitochondrial iron-sulfur protein biogenesis.
      ) (Fig. 3B). Domains 1 (residues 90–192) and 2 (residues 73–89 and 195–248) are separated from domain 3 (residues 310–422) by three α-helices that wrap the entire protein (α4, α6, and α9). Superposition analysis with the structures of the human ortholog HsIBA57 (PDB code: 6QE4), the E. coli homolog EcYgfZ (PDB code: 1NRK), and the T-subunit HsGcvT (aka GCST) of the human mitochondrial glycine cleavage complex (PDB code: 1WSV) showed an overall low rmsd of 1.012 over 204 Cα atoms, 1.106 over 120 Cα atoms, and 1.230 over 87 Cα atoms, respectively (Fig. 3C). Despite the overall low primary sequence conservation, the structures of the two mitochondrial Iba57 proteins are virtually identical, except for a short 25-residue long insertion present in CtIba57 (and other fungal relatives) in front of the conserved C-terminal PxW motif (Fig. S3A). Moreover, and of importance for our further study, the core regions of the Iba57 proteins display a high structural similarity with both EcYgfZ and HsGcvT.
      Figure thumbnail gr3
      Figure 3Crystal structure of Chaetomium thermophilum Iba57 at 2.4 Å resolution. A, disulfide-bridged (via Cys304) dimer of CtIba57 as found in the crystal. Residues are rainbow colored from N (blue) to C termini (red). B, cartoon representation of CtIba57 monomer (coloring as in A). The three individual domains are highlighted in gray (
      • Zanello P.
      Structure and electrochemistry of proteins harboring iron-sulfur clusters of different nuclearities. Part IV. Canonical, non-canonical and hybrid iron-sulfur proteins.
      ,
      • Braymer J.J.
      • Freibert S.A.
      • Rakwalska-Bange M.
      • Lill R.
      Mechanistic concepts of iron-sulfur protein biogenesis in biology.
      ,
      • Lill R.
      • Freibert S.A.
      Mechanisms of mitochondrial iron-sulfur protein biogenesis.
      ). The sulfur of the essential Cys304 is depicted as a yellow sphere. C, superposition of CtIba57 (green) with HsIBA57 (blue, left; Protein Data Bank [PDB] code: 6QE4), EcYgfZ (gray, middle; PDB code: 1NRK), and HsGcvT (magenta, right; PDB code: 1WSV). The conserved Cys residues of Iba57 (Ct and Hs) and EcYgfZ as well as the structural equivalent Asp273 of HsGcvT are depicted as spheres. THF of HsGcvT is shown in yellow (for enlargement, see A). CtIba57, CtIba57, Chaetomium thermophilum Iba57; EcYgfZ, Escherichia coli YgfZ; HsGcvT, human GcvT; HsIBA57, human IBA57; THF, tetrahydrofolate.
      Reminiscent of the EcYgfZ and HsIBA57 structures, we did not detect any THF or related folates within the crystallized CtIba57. To gain structural insights into potential THF binding to Iba57 and YgfZ proteins, we compared the THF-binding region of HsGcvT with the respective areas in the Iba57–YgfZ structures (
      • Okamura-Ikeda K.
      • Hosaka H.
      • Yoshimura M.
      • Yamashita E.
      • Toma S.
      • Nakagawa A.
      • et al.
      Crystal structure of human T-protein of glycine cleavage system at 2.0 A resolution and its implication for understanding non-ketotic hyperglycinemia.
      ). The glycine cleavage complex catalyzes the oxidative decarboxylation of glycine required for the formation of 5,10-methylene-THF (
      • Okamura-Ikeda K.
      • Hosaka H.
      • Maita N.
      • Fujiwara K.
      • Yoshizawa A.C.
      • Nakagawa A.
      • et al.
      Crystal structure of aminomethyltransferase in complex with dihydrolipoyl-H-protein of the glycine cleavage system: Implications for recognition of lipoyl protein substrate, disease-related mutations, and reaction mechanism.
      ,
      • Kikuchi G.
      • Motokawa Y.
      • Yoshida T.
      • Hiraga K.
      Glycine cleavage system: Reaction mechanism, physiological significance, and hyperglycinemia.
      ). Its T-subunit GcvT is a THF-dependent amino methyltransferase that catalyzes the transfer of a methylene one-carbon unit to THF from a methylamine group at the lipoyl arm of the H-subunit GCSH of the glycine cleavage complex. In GcvT enzymes, THF binds in a central binding cleft formed by the two N-terminal domains of the protein with the pteridine ring being buried in a hydrophobic pocket and its glutamyl group pointing outward (Fig. 3C, right; enlargement in Fig. 4A) (
      • Okamura-Ikeda K.
      • Hosaka H.
      • Yoshimura M.
      • Yamashita E.
      • Toma S.
      • Nakagawa A.
      • et al.
      Crystal structure of human T-protein of glycine cleavage system at 2.0 A resolution and its implication for understanding non-ketotic hyperglycinemia.
      ,
      • Lokanath N.K.
      • Kuroishi C.
      • Okazaki N.
      • Kunishima N.
      Crystal structure of a component of glycine cleavage system: T-Protein from pyrococcus horikoshii OT3 at 1.5 A resolution.
      ,
      • Lee H.H.
      • Kim D.J.
      • Ahn H.J.
      • Ha J.Y.
      • Suh S.W.
      Crystal structure of T-protein of the glycine cleavage system. Cofactor binding, insights into H-protein recognition, and molecular basis for understanding nonketotic hyperglycinemia.
      ,
      • Orun O.
      • Koch M.H.
      • Kan B.
      • Svergun D.I.
      • Petoukhov M.V.
      • Sayers Z.
      Structural characterization of T-protein of the Escherichia coli glycine cleavage system by X-ray small angle scattering.
      ). Superposition of HsGcvT and CtIba57 shows that the CtIba57 loop connecting β9 and β10 strands from domain 2 (β9–10-loop) and the loop adjacent to α5 (α5-loop) are shifted toward each other (Fig. 4A; blue arrows). As a consequence, the potential entry tunnel to the pteridine pocket is virtually closed in CtIba57. This scenario also holds true for HsIBA57 and for the α5-loop of EcYgfZ (Fig. S4). In addition, the entire THF-binding pocket as found in HsGcvT is almost completely filled in Iba57 proteins, because the side chains of the two loops protrude into this pocket (Figs. 4B and 5, A and B).
      Figure thumbnail gr4
      Figure 4The structure of CtIba57 is incompatible with THF binding. A, superposition of CtIba57 (green) and HsGcvT (magenta; Protein Data Bank [PDB] code: 1WSV) highlighting the THF (yellow)-binding region of HsGcvT. The loops β9–10 and α5 that would interfere with THF binding in CtIba57 are marked. Blue arrows indicate the distances of the structural rearrangements of the respective loops. (For a comparison including HsIBA57 and EcYgfZ, refer to ). B, the cavity of HsGcvT (red) encompassing its THF-binding site (THF pocket) and its lipoyl entrance tunnel was calculated using PyMOL. Side chains of residues in β9–10 and α5 loops of CtIba57 (green; shown as balls and sticks) protrude into the THF pocket of HsGcvT. C, comparison of residues involved in THF (yellow) binding to various proteins. Left, the canonical THF-binding pocket as found in HsGcvT (magenta), TrmE (cyan; PDB code: 1XZQ), and DMGO (sienna; PDB code: 1PJ6). The residue numbers are indicated in parenthesis for HsGcvT. Right, comparison of the THF-binding residues of HsGcvT (as shown left) with the structurally equivalent residues of CtIba57 (green), HsIBA57 (blue, PDB code: 6QE4), and EcYgfZ (gray, PDB code: 1NRK). The residue numbers are indicated in parenthesis for CtIba57. Only the side chains are depicted as sticks. Contacts are indicated by black lines. The catalytically important N10 position of THF is marked. CtIba57, Chaetomium thermophilum Iba57; EcYgfZ, Escherichia coli YgfZ; HsGcvT, human GcvT; HsIBA57, human IBA57; THF, tetrahydrofolate.
      Figure thumbnail gr5
      Figure 5Cavities and surfaces of Iba57 and EcYgfZ exclude productive THF binding. A, cavities inside the cartoon ribbon representation were calculated for the indicated proteins using PyMOL. In HsGcvT, the cavity forms both the tunnel for the lipoyl arm of GCSH and the binding pocket for THF (yellow). B, the cavities extracted from A with attached human GCSH protein (location taken from the GCSH–HsGcvT complex (Protein Data Bank [PDB] code: 3A8I). The dihydrolipoyl moiety attached to GCSH protein is depicted in light green. THF (yellow) is shown as found in HsGcvT. C, surface charges (calculated by APBS biomolecular solvation software suite (
      • Jurrus E.
      • Engel D.
      • Star K.
      • Monson K.
      • Brandi J.
      • Felberg L.E.
      • et al.
      Improvements to the APBS biomolecular solvation software suite.
      )) of the indicated proteins (blue: positive; red, negative). The β9–10- and α5-loops and the C terminus are indicated in CtIba57. The potential THF-binding pocket is indicated in EcYgfZ (yellow arrow). THF in HsGcvT is shown in yellow, and the poly-Glu binding site is outlined by a yellow dotted line. CtIba57, Chaetomium thermophilum Iba57; EcYgfZ, Escherichia coli YgfZ; HsGcvT, human GcvT; THF, tetrahydrofolate.
      In HsGcvT, several residues are involved in THF binding. In particular, Met56, Asp101, Tyr197, Glu204, and R233 of HsGcvT (PDB code: 1WSV; residues highlighted in yellow in Fig. S3) make contacts with THF. The first four residues are invariant in THF-binding family proteins such as HsGcvT homologs, the guanine nucleotide-binding protein TrmE (PDB code: 1XZQ), and dimethylglycine oxidase DMGO (PDB code: 1PJ6), and define the canonical THF-binding residues (
      • Okamura-Ikeda K.
      • Hosaka H.
      • Yoshimura M.
      • Yamashita E.
      • Toma S.
      • Nakagawa A.
      • et al.
      Crystal structure of human T-protein of glycine cleavage system at 2.0 A resolution and its implication for understanding non-ketotic hyperglycinemia.
      ,
      • Scrutton N.S.
      • Leys D.
      Crystal structure of DMGO provides a prototype for a new tetrahydrofolate-binding fold.
      ,
      • Leys D.
      • Basran J.
      • Scrutton N.S.
      Channelling and formation of 'active' formaldehyde in dimethylglycine oxidase.
      ,
      • Scrima A.
      • Vetter I.R.
      • Armengod M.E.
      • Wittinghofer A.
      The structure of the TrmE GTP-binding protein and its implications for tRNA modification.
      ) (Fig. 4C, left panel). R233 is specific for GcvT proteins. While in HsGcvT, Met56 and Tyr197 form the hydrophobic pocket for the pteridine group, the side chain of Glu204 undergoes a double hydrogen bonding toward the amino group bound to the C2 position and the N3 of the pteridine ring (
      • Okamura-Ikeda K.
      • Hosaka H.
      • Yoshimura M.
      • Yamashita E.
      • Toma S.
      • Nakagawa A.
      • et al.
      Crystal structure of human T-protein of glycine cleavage system at 2.0 A resolution and its implication for understanding non-ketotic hyperglycinemia.
      ,
      • Lokanath N.K.
      • Kuroishi C.
      • Okazaki N.
      • Kunishima N.
      Crystal structure of a component of glycine cleavage system: T-Protein from pyrococcus horikoshii OT3 at 1.5 A resolution.
      ,
      • Lee H.H.
      • Kim D.J.
      • Ahn H.J.
      • Ha J.Y.
      • Suh S.W.
      Crystal structure of T-protein of the glycine cleavage system. Cofactor binding, insights into H-protein recognition, and molecular basis for understanding nonketotic hyperglycinemia.
      ). In one-carbon transfer, Asp101 assists the nucleophilic attack of the catalytic N10 group of THF on the methylene carbon on the GCSH-bound lipoyl arm by proton abstraction (
      • Kikuchi G.
      • Motokawa Y.
      • Yoshida T.
      • Hiraga K.
      Glycine cleavage system: Reaction mechanism, physiological significance, and hyperglycinemia.
      ,
      • Scrutton N.S.
      • Leys D.
      Crystal structure of DMGO provides a prototype for a new tetrahydrofolate-binding fold.
      ,
      • Scrima A.
      • Vetter I.R.
      • Armengod M.E.
      • Wittinghofer A.
      The structure of the TrmE GTP-binding protein and its implications for tRNA modification.
      ).
      In CtIba57, the positively charged Arg90 replaces the hydrophobic Met56 of HsGcvT (residues Arg56 in HsIBA57 and Trp27 in EcYgfZ), and the position occupied by Tyr197 in HsGcvT does not exist, neither in any of the Iba57 proteins nor in EcYgfZ (Fig. 4C, right panel; Fig. S3, highlighted in yellow). These differences fully abolished the hydrophobic cavity accommodating the pteridine group of THF in HsGcvT. The negatively charged Glu204 in HsGcvT is replaced by hydrophobic residues in CtIba57 (Ile236), HsIBA57 (Leu198), and EcYgfZ (Ile163), thus preventing hydrogen bonding to the pteridine ring. Interestingly, the invariant and catalytically important residue Asp101 of HsGcvT was retained in CtIba57 (Asp141), HsIBA57 (Asp109), and in most bacterial YgfZ proteins (Fig. 4C, right panel; Fig. S3, highlighted in yellow). As an exception to most bacterial YgfZ members, EcYgfZ carries an Asn at this position (Asn72).
      We finally investigated the crystal structures of CtIba57, HsIBA57, and EcYgfZ for the presence of potential cavities as seen in HsGcvT for the lipoyl entry tunnel and the THF-binding pocket (calculated by PyMOL 2.0; Schrödinger, LLC). We found significant differences (Fig. 5, A and B). While the mitochondrial Iba57 proteins completely lack the THF-binding pocket (Fig. 4B), they retain the entry tunnel of HsGcvT for the lipoyl arm of the GCSH subunit of the glycine cleavage system, although the respective residues are not conserved (Fig. S3B). Moreover, HsIBA57 and CtIba57 completely lack the positively charged patch located at the C terminus of HsGcvT that binds the polyglutamate tail of THF (
      • Okamura-Ikeda K.
      • Hosaka H.
      • Yoshimura M.
      • Yamashita E.
      • Toma S.
      • Nakagawa A.
      • et al.
      Crystal structure of human T-protein of glycine cleavage system at 2.0 A resolution and its implication for understanding non-ketotic hyperglycinemia.
      ,
      • Lokanath N.K.
      • Kuroishi C.
      • Okazaki N.
      • Kunishima N.
      Crystal structure of a component of glycine cleavage system: T-Protein from pyrococcus horikoshii OT3 at 1.5 A resolution.
      ,
      • Lee H.H.
      • Kim D.J.
      • Ahn H.J.
      • Ha J.Y.
      • Suh S.W.
      Crystal structure of T-protein of the glycine cleavage system. Cofactor binding, insights into H-protein recognition, and molecular basis for understanding nonketotic hyperglycinemia.
      ,
      • Scrima A.
      • Vetter I.R.
      • Armengod M.E.
      • Wittinghofer A.
      The structure of the TrmE GTP-binding protein and its implications for tRNA modification.
      ) (Fig. 5C). In eukaryotes, folylpolyglutamylation significantly enhances the THF affinity for mitochondrial and cytosolic folate–dependent enzymes and is required for folate retention within the cell, particularly in mitochondria (
      • Cherest H.
      • Thomas D.
      • Surdin-Kerjan Y.
      Polyglutamylation of folate coenzymes is necessary for methionine biosynthesis and maintenance of intact mitochondrial genome in Saccharomyces cerevisiae.
      ,
      • Raz S.
      • Stark M.
      • Assaraf Y.G.
      Folylpoly-gamma-glutamate synthetase: a key determinant of folate homeostasis and antifolate resistance in cancer.
      ,
      • Osborne C.B.
      • Lowe K.E.
      • Shane B.
      Regulation of folate and one-carbon metabolism in mammalian-cells .1. Folate metabolism in Chinese-hamster ovary cells expressing escherichia-coli or human folylpoly-gamma-glutamate synthetase-activity.
      ). Collectively, these structural considerations strongly suggest that mitochondrial Iba57 proteins appear to be unable to bind and utilize THF for enzymatic function. The situation seems only slightly different for EcYgfZ, where the THF-binding pocket appears to be partially retained, yet with an amino acid composition differing significantly from the canonical folate-binding site (Fig. 4C). Furthermore, the potential entrance for the lipoyl arm of GCSH is fully blocked in EcYgfZ, and the positively charged polyglutamate-binding patch of HsGcvT is poorly maintained. These substantial structural changes of EcYgfZ relative to HsGcvT explain well why only a low affinity binding of folates (millimolar range) was observed for EcYgfZ (
      • Waller J.C.
      • Alvarez S.
      • Naponelli V.
      • Lara-Nunez A.
      • Blaby I.K.
      • Da Silva V.
      • et al.
      A role for tetrahydrofolates in the metabolism of iron-sulfur clusters in all domains of life.
      ) (see also Discussion section). Taken together, the low sequence conservation and, more importantly, the drastic changes in the topology and biochemical environment of the HsGcvT-related THF-binding area render physiologically relevant THF binding to mitochondrial Iba57 proteins and in turn a THF-dependent function unlikely. Based on similar, yet characteristically different structural considerations, the same appears to be true for EcYgfZ.

      Mutational analyses confirm the THF-independent function of Iba57

      One of the few positions of primary sequence conservation between the Iba57–YgfZ and GcvT protein families is the well-conserved Asp141 of CtIba57 that within HsGcvT (Asp101) assists the nucleophilic attack of the reactive N10 group of THF on the methylene carbon on the lipoyl arm (Fig. 4C; Fig. S3) (
      • Kikuchi G.
      • Motokawa Y.
      • Yoshida T.
      • Hiraga K.
      Glycine cleavage system: Reaction mechanism, physiological significance, and hyperglycinemia.
      ,
      • Scrutton N.S.
      • Leys D.
      Crystal structure of DMGO provides a prototype for a new tetrahydrofolate-binding fold.
      ,
      • Scrima A.
      • Vetter I.R.
      • Armengod M.E.
      • Wittinghofer A.
      The structure of the TrmE GTP-binding protein and its implications for tRNA modification.
      ). In HsGcvT, mutations of Asp101 to Asn or Ala are associated with a complete loss of function (
      • Okamura-Ikeda K.
      • Hosaka H.
      • Yoshimura M.
      • Yamashita E.
      • Toma S.
      • Nakagawa A.
      • et al.
      Crystal structure of human T-protein of glycine cleavage system at 2.0 A resolution and its implication for understanding non-ketotic hyperglycinemia.
      ). As mentioned previously, in EcYgfZ, this residue is changed to Asn, further challenging the claim of a THF-dependent enzymatic function of EcYgfZ. Since the catalytic Asp101 of HsGcvT is conserved in mitochondrial Iba57 proteins, it serves as an excellent residue to test for potential THF-dependent catalytic function of Iba57. We employed yeast genetics and exchanged the corresponding Asp149 of S. cerevisiae Iba57 to Asn or Ala (cf. Fig. S3A). We further substituted residue Arg376, which is invariant in mitochondrial Iba57 proteins by His (Fig. S5). In HsGcvT, the corresponding residue Arg292 binds to the α-carboxylate group of THF and is conserved in eukaryotes. Its substitution by His results in decreased affinity to THF, loss of enzymatic activity, and causes nonketotic hyperglycinemia (
      • Zheng Y.
      • Cantley L.C.
      Toward a better understanding of folate metabolism in health and disease.
      ,
      • Okamura-Ikeda K.
      • Hosaka H.
      • Yoshimura M.
      • Yamashita E.
      • Toma S.
      • Nakagawa A.
      • et al.
      Crystal structure of human T-protein of glycine cleavage system at 2.0 A resolution and its implication for understanding non-ketotic hyperglycinemia.
      ,
      • Toone J.R.
      • Applegarth D.A.
      • Levy H.L.
      • Coulter-Mackie M.B.
      • Lee G.
      Molecular genetic and potential biochemical characteristics of patients with T-protein deficiency as a cause of glycine encephalopathy (NKH).
      ). Furthermore, we created a yeast mutant in which residues Arg366 and Arg374 of ScIba57 (Arg301 und Arg309 in CtIba57) were changed to Ala (termed R1,R2A). These residues are conserved in the Iba57–YgfZ family and might potentially contribute to a positively charged surface patch that in HsGcvT interacts with the polyglutamate moiety of folylpolyglutamate, thereby stabilizing THF binding to HsGcvT (
      • Zheng Y.
      • Cantley L.C.
      Toward a better understanding of folate metabolism in health and disease.
      ,
      • Scrima A.
      • Vetter I.R.
      • Armengod M.E.
      • Wittinghofer A.
      The structure of the TrmE GTP-binding protein and its implications for tRNA modification.
      ,
      • Raz S.
      • Stark M.
      • Assaraf Y.G.
      Folylpoly-gamma-glutamate synthetase: a key determinant of folate homeostasis and antifolate resistance in cancer.
      ,
      • Lawrence S.A.
      • Titus S.A
      • Ferguson J.
      • Heineman A.L.
      • Taylor S.M.
      • Moran R.G.
      Mammalian mitochondrial and cytosolic folylpolyglutamate synthetase maintain the subcellular compartmentalization of folates.
      ) (Fig. 5C and S3A). The corresponding ScIba57 variants were expressed from low-copy plasmids under the control of the endogenous promoter, and all perfectly rescued the Lys and Glu auxotrophy of iba57Δ yeast cells (Fig. 6A). As a control, we analyzed the in vivo consequences of exchanging the conserved surface-exposed Cys357 of the KGC(Y/F)XGQEL signature motif present in both mitochondrial Iba57 and bacterial YgfZ proteins (Fig. S3A). In E. coli, this residue is essential for EcYgfZ function in vivo (
      • Hasnain G.
      • Waller J.C.
      • Alvarez S.
      • Ravilious G.E.
      • Jez J.M.
      • Hanson A.D.
      Mutational analysis of YgfZ, a folate-dependent protein implicated in iron/sulphur cluster metabolism.
      ,
      • Lin C.N.
      • Syu W.J.
      • Sun W.S.
      • Chen J.W.
      • Chen T.H.
      • Don M.J.
      • et al.
      A role of ygfZ in the Escherichia coli response to plumbagin challenge.
      ). Biochemical studies have shown a crucial function of this residue in HsIBA57 during the in vitro synthesis of [4Fe–4S] clusters (
      • Weiler B.D.
      • Bruck M.C.
      • Kothe I.
      • Bill E.
      • Lill R.
      • Muhlenhoff U.
      Mitochondrial [4Fe-4S] protein assembly involves reductive [2Fe-2S] cluster fusion on ISCA1-ISCA2 by electron flow from ferredoxin FDX2.
      ). As expected from these studies, ScIba57–C357A and ScIba57–C357S variants were unable to rescue the Lys and Glu auxotrophy of iba57Δ cells, when expressed from low-copy plasmids under the control of the endogenous promoter (Fig. 6A), distinguishing this strong phenotype from the inconspicuous behavior of potential THF-binding residues of ScIba57.
      Figure thumbnail gr6
      Figure 6Potential THF-interacting residues are dispensable for in vivo function of ScIba57. A, yeast iba57Δ cells expressing either wildtype (WT) ScIba57 or the indicated point mutation variants from a centromeric plasmid (p416) under the control of the endogenous IBA57 promoter were cultivated on solid synthetic defined (SD) minimal medium in the presence or the absence of Lys and Glu. iba57Δ with an empty vector (416) served as control. Overproduction of the ScIba57–C357S variant in iba57Δ cells from a high-copy vector (p426) did not lead to high-copy suppression. B and C, cells cultivated in SD minimal medium plus Lys and Glu were assayed for aconitase enzyme activities (relative to malate dehydrogenase [MDH]). D and E, the presence of aconitase and the lipoylation (LA) of the E2 subunits of pyruvate (Lat1) and 2-ketoglutarate dehydrogenases (Kgd2) was determined by immunostaining of isolated mitochondrial extracts. A stain for mitochondrial Por1 served as a loading control. Error bars indicate the standard deviation (n ≥ 4). ScIba57, Saccharomyces cerevisiae Iba57; THF, tetrahydrofolate.
      We further analyzed the aforementioned mutants biochemically. In keeping with the growth phenotypes, iba57Δ cells expressing the potential THF-related ScIba57 variants showed wildtype aconitase activities, and the lipoylation of mitochondrial pyruvate and α-ketoglutarate dehydrogenase subunits was restored to wildtype levels (Fig. 6, BE). In contrast, the two ScIba57–C357 variants did not show any aconitase or lipoylation activities. The same was seen when the C357S mutant was overexpressed from the strong TDH3 promoter in a high-copy vector. This lack of high-copy suppression demonstrates that residue Cys357 is fully indispensable for in vivo function. Together, these physiological and biochemical findings clearly rule out a THF-dependent catalytic function of ScIba57 and thus fully support the conclusions drawn from inspection of the two Iba57 crystal structures.

      Discussion

      In eukaryotes, the late-acting ISC factors Isa1, Isa2, and Iba57 play an essential role in mitochondrial [4Fe–4S] protein biogenesis, and their function may in principle be conserved in bacteria (
      • Gelling C.
      • Dawes I.W.
      • Richhardt N.
      • Lill R.
      • Muhlenhoff U.
      Mitochondrial Iba57p is required for Fe/S cluster formation on aconitase and activation of radical SAM enzymes.
      ,
      • Muhlenhoff U.
      • Richter N.
      • Pines O.
      • Pierik A.J.
      • Lill R.
      Specialized function of yeast Isa1 and Isa2 proteins in the maturation of mitochondrial [4Fe-4S] proteins.
      ,
      • Long S.
      • Changmai P.
      • Tsaousis A.D.
      • Skalicky T.
      • Verner Z.
      • Wen Y.Z.
      • et al.
      Stage-specific requirement for Isa1 and Isa2 proteins in the mitochondrion of Trypanosoma brucei and heterologous rescue by human and blastocystis orthologues.
      ,
      • Sheftel A.D.
      • Wilbrecht C.
      • Stehling O.
      • Niggemeyer B.
      • Elsasser H.P.
      • Muhlenhoff U.
      • et al.
      The human mitochondrial ISCA1, ISCA2, and IBA57 proteins are required for [4Fe-4S] protein maturation.
      ,
      • Py B.
      • Gerez C.
      • Huguenot A.
      • Vidaud C.
      • Fontecave M.
      • Ollagnier de Choudens S.
      • et al.
      The ErpA/NfuA complex builds an oxidation-resistant Fe-S cluster delivery pathway.
      ). In tight cooperation, they catalyze the reductive fusion of [2Fe–2S] clusters provided by the early parts of the mitochondrial ISC system to a [4Fe–4S] cluster (
      • Weiler B.D.
      • Bruck M.C.
      • Kothe I.
      • Bill E.
      • Lill R.
      • Muhlenhoff U.
      Mitochondrial [4Fe-4S] protein assembly involves reductive [2Fe-2S] cluster fusion on ISCA1-ISCA2 by electron flow from ferredoxin FDX2.
      ). The interplay of these three proteins and the precise mechanistic role of each of these mitochondrial ISC factors are poorly resolved to date. Iba57 and its bacterial homolog YgfZ belong to the COG0354 protein family of folate-binding proteins with structural similarities to the T-subunit GcvT of the glycine cleavage system (
      • Gourdoupis S.
      • Nasta V.
      • Calderone V.
      • Ciofi-Baffoni S.
      • Banci L.
      IBA57 recruits ISCA2 to form a [2Fe-2S] cluster-mediated complex.
      ,
      • Teplyakov A.
      • Obmolova G.
      • Sarikaya E.
      • Pullalarevu S.
      • Krajewski W.
      • Galkin A.
      • et al.
      Crystal structure of the YgfZ protein from Escherichia coli suggests a folate-dependent regulatory role in one-carbon metabolism.
      ). On first glance, this suggests a THF-dependent enzymatic function for both Iba57 and YgfZ. Consistent with this idea, E. coli strains with defects in folate biosynthesis or with a deletion of ygfZ show a 40% lower activity of MiaB, a radical SAM [4Fe–4S] protein involved in tRNA modification (
      • Waller J.C.
      • Alvarez S.
      • Naponelli V.
      • Lara-Nunez A.
      • Blaby I.K.
      • Da Silva V.
      • et al.
      A role for tetrahydrofolates in the metabolism of iron-sulfur clusters in all domains of life.
      ,
      • Hasnain G.
      • Waller J.C.
      • Alvarez S.
      • Ravilious G.E.
      • Jez J.M.
      • Hanson A.D.
      Mutational analysis of YgfZ, a folate-dependent protein implicated in iron/sulphur cluster metabolism.
      ,
      • Waller J.C.
      • Ellens K.W.
      • Alvarez S.
      • Loizeau K.
      • Ravanel S.
      • Hanson A.D.
      Mitochondrial and plastidial COG0354 proteins have folate-dependent functions in iron-sulphur cluster metabolism.
      ). Moreover, EcYgfZ binds folate derivatives in vitro, yet with rather low (millimolar range) affinity. Since EcYgfZ can be functionally replaced in E. coli by mitochondrial Iba57 from various organisms including yeast and plants, it was proposed that the supposed folate-requiring function of EcYgfZ in the biogenesis of [4Fe–4S] proteins is generally conserved in mitochondrial Iba57 relatives. Yet, our genetic, mutational, biochemical, and structural studies refute a folate-dependent function of Iba57 (for discussion of YgfZ, see later). We provide three independent lines of evidence for this conclusion: (i) the lack of folate requirement for mitochondrial [4Fe–4S] protein maturation, (ii) the absence of a characteristic THF-binding pocket in Iba57 structures, and (iii) the inconspicuous phenotype of mutations of conserved Iba57 residues that are important for THF binding and THF-dependent catalysis in GcvT.
      Our investigations of S. cerevisiae folate synthesis mutants (that can be complemented by the four folate-requiring metabolites, that is, adenine, His, Met, and dTMP (
      • Bayly A.M.
      • Berglez J.M.
      • Patel O.
      • Castelli L.A.
      • Hankins E.G.
      • Coloe P.
      • et al.
      Folic acid utilisation related to sulfa drug resistance in Saccharomyces cerevisiae.
      ,
      • Christensen K.E.
      • MacKenzie R.E.
      Mitochondrial one-carbon metabolism is adapted to the specific needs of yeast, plants and mammals.
      ,
      • Locasale J.W.
      Serine, glycine and one-carbon units: Cancer metabolism in full circle.
      )), revealed no folate requirement for the activities of mitochondrial [4Fe–4S] enzymes such as aconitase and lipoyl synthase, two key mitochondrial Fe–S proteins whose maturation strictly depends on Iba57 (
      • Gelling C.
      • Dawes I.W.
      • Richhardt N.
      • Lill R.
      • Muhlenhoff U.
      Mitochondrial Iba57p is required for Fe/S cluster formation on aconitase and activation of radical SAM enzymes.
      ). This finding suggested no general involvement of folate in Iba57 function. Moreover, our folate synthesis mutants showed no auxotrophies for the two amino acids Lys and Glu that require mitochondrial [4Fe–4S] aconitase and homoaconitase for their synthesis. These requirements are characteristic genetic hallmarks of yeast cells deficient in Iba57, Isa1, or Isa2 (
      • Gelling C.
      • Dawes I.W.
      • Richhardt N.
      • Lill R.
      • Muhlenhoff U.
      Mitochondrial Iba57p is required for Fe/S cluster formation on aconitase and activation of radical SAM enzymes.
      ,
      • Muhlenhoff U.
      • Richter N.
      • Pines O.
      • Pierik A.J.
      • Lill R.
      Specialized function of yeast Isa1 and Isa2 proteins in the maturation of mitochondrial [4Fe-4S] proteins.
      ). Furthermore, our observations are fully consistent with earlier studies on folate-deficient yeast mutants that did not document any Lys or Glu requirements (
      • Zheng Y.
      • Cantley L.C.
      Toward a better understanding of folate metabolism in health and disease.
      ,
      • Bayly A.M.
      • Berglez J.M.
      • Patel O.
      • Castelli L.A.
      • Hankins E.G.
      • Coloe P.
      • et al.
      Folic acid utilisation related to sulfa drug resistance in Saccharomyces cerevisiae.
      ,
      • Christensen K.E.
      • MacKenzie R.E.
      Mitochondrial one-carbon metabolism is adapted to the specific needs of yeast, plants and mammals.
      ,
      • Locasale J.W.
      Serine, glycine and one-carbon units: Cancer metabolism in full circle.
      ,
      • Cherest H.
      • Thomas D.
      • Surdin-Kerjan Y.
      Polyglutamylation of folate coenzymes is necessary for methionine biosynthesis and maintenance of intact mitochondrial genome in Saccharomyces cerevisiae.
      ). Hence, our conclusion that folate does not play a role in mitochondrial [4Fe–4S] protein metabolism is fully in line with the available knowledge of folate and one-carbon metabolism in S. cerevisiae.
      The crystal structures of both HsIBA57 and C. thermophilum Iba57 display an overall high structural similarity to THF-binding proteins including HsGcvT of the glycine cleavage system. Nevertheless, closer inspection of the Iba57 structures revealed several important differences that are incompatible with THF binding to Iba57. In both Iba57 proteins, the THF entry tunnel is blocked by two loops that have moved substantially from their original position in HsGcvT toward each other in Iba57. Furthermore, the THF-binding pocket is constricted and appears too small for accommodation of a folate molecule. More importantly, key residues that specifically coordinate the THF molecule in the HsGcvT-binding pocket are either absent in the Iba57 structures or are exchanged from charged to hydrophobic amino acids and vice versa. In Iba57, these nonconservative amino acid replacements are frequently shifted in position and point slightly away from the putative THF partner. As the only notable exception, the catalytically essential Asp101 of the THF-binding pocket of HsGcvT is fully conserved in Iba57 proteins (
      • Okamura-Ikeda K.
      • Hosaka H.
      • Yoshimura M.
      • Yamashita E.
      • Toma S.
      • Nakagawa A.
      • et al.
      Crystal structure of human T-protein of glycine cleavage system at 2.0 A resolution and its implication for understanding non-ketotic hyperglycinemia.
      ,
      • Lee H.H.
      • Kim D.J.
      • Ahn H.J.
      • Ha J.Y.
      • Suh S.W.
      Crystal structure of T-protein of the glycine cleavage system. Cofactor binding, insights into H-protein recognition, and molecular basis for understanding nonketotic hyperglycinemia.
      ), yet, as discussed later, is not essential for yeast Iba57 function. Finally, the basic surface area that binds the polyglutamyl tail of THF and enhances THF binding in GcvT proteins is virtually absent in mitochondrial Iba57 structures. In eukaryotes, this tail is required for retention of THF in mitochondria and cytosol (
      • Cherest H.
      • Thomas D.
      • Surdin-Kerjan Y.
      Polyglutamylation of folate coenzymes is necessary for methionine biosynthesis and maintenance of intact mitochondrial genome in Saccharomyces cerevisiae.
      ,
      • Raz S.
      • Stark M.
      • Assaraf Y.G.
      Folylpoly-gamma-glutamate synthetase: a key determinant of folate homeostasis and antifolate resistance in cancer.
      ,
      • Lawrence S.A.
      • Titus S.A
      • Ferguson J.
      • Heineman A.L.
      • Taylor S.M.
      • Moran R.G.
      Mammalian mitochondrial and cytosolic folylpolyglutamate synthetase maintain the subcellular compartmentalization of folates.
      ). In contrast, the lipoyl tunnel of GcvT that accommodates this cofactor attached to the GCSH subunit of the glycine-cleavage complex is maintained in Iba57 (
      • Okamura-Ikeda K.
      • Hosaka H.
      • Maita N.
      • Fujiwara K.
      • Yoshizawa A.C.
      • Nakagawa A.
      • et al.
      Crystal structure of aminomethyltransferase in complex with dihydrolipoyl-H-protein of the glycine cleavage system: Implications for recognition of lipoyl protein substrate, disease-related mutations, and reaction mechanism.
      ), yet many of the residues lining the tunnel are exchanged, rendering lipoyl binding unlikely. Consistently, no interaction between GCSH and Iba57 proteins is known (see, e.g., (
      • Beilschmidt L.K.
      • Ollagnier de Choudens S.
      • Fournier M.
      • Sanakis I.
      • Hograindleur M.A.
      • Clemancey M.
      • et al.
      ISCA1 is essential for mitochondrial Fe4S4 biogenesis in vivo.
      )). Taken together, our structural analyses indicate that mitochondrial Iba57 has evolved into a THF-independent protein by altering the canonical THF-binding cavity of common GcvT-like precursors into a filled region. As a result, the Iba57 structures strongly argue against a THF-dependent function in mitochondrial [4Fe–4S] cluster formation. The functional role of the remnant THF domain, if any, remains to be determined.
      A THF-independent function of mitochondrial Iba57 proteins is further made unlikely by mutational studies in vivo. THF-dependent one-carbon transfer reactions catalyzed by amino-methyltransferase GcvT (
      • Okamura-Ikeda K.
      • Hosaka H.
      • Yoshimura M.
      • Yamashita E.
      • Toma S.
      • Nakagawa A.
      • et al.
      Crystal structure of human T-protein of glycine cleavage system at 2.0 A resolution and its implication for understanding non-ketotic hyperglycinemia.
      ,
      • Lokanath N.K.
      • Kuroishi C.
      • Okazaki N.
      • Kunishima N.
      Crystal structure of a component of glycine cleavage system: T-Protein from pyrococcus horikoshii OT3 at 1.5 A resolution.
      ,
      • Lee H.H.
      • Kim D.J.
      • Ahn H.J.
      • Ha J.Y.
      • Suh S.W.
      Crystal structure of T-protein of the glycine cleavage system. Cofactor binding, insights into H-protein recognition, and molecular basis for understanding nonketotic hyperglycinemia.
      ), dimethylglycine oxidase DMGO (
      • Leys D.
      • Basran J.
      • Scrutton N.S.
      Channelling and formation of 'active' formaldehyde in dimethylglycine oxidase.
      ), or guanine nucleotide-binding protein TrmE (
      • Scrima A.
      • Vetter I.R.
      • Armengod M.E.
      • Wittinghofer A.
      The structure of the TrmE GTP-binding protein and its implications for tRNA modification.
      ) essentially involve a strictly conserved aspartate residue (Asp101 in HsGcvT) that transiently forms a hydrogen bond with the catalytically important N10 group of THF. Asp101 assists the nucleophilic attack of the catalytic N10 group of THF on the methylene carbon on the lipoyl arm by proton abstraction. The corresponding aspartate in mitochondrial Iba57 is fully conserved. Yet, while exchanges of this residue in HsGcvT result in complete loss of enzymatic function in vitro (
      • Okamura-Ikeda K.
      • Hosaka H.
      • Yoshimura M.
      • Yamashita E.
      • Toma S.
      • Nakagawa A.
      • et al.
      Crystal structure of human T-protein of glycine cleavage system at 2.0 A resolution and its implication for understanding non-ketotic hyperglycinemia.
      ), this residue is dispensable for Iba57 function in vivo, as indicated by inconspicuous growth phenotypes and wildtype Fe–S protein maturation activities. Furthermore, several other residues, which in HsGcvT interact with THF and upon exchange impair its function (
      • Okamura-Ikeda K.
      • Hosaka H.
      • Yoshimura M.
      • Yamashita E.
      • Toma S.
      • Nakagawa A.
      • et al.
      Crystal structure of human T-protein of glycine cleavage system at 2.0 A resolution and its implication for understanding non-ketotic hyperglycinemia.
      ), are nonessential for Iba57 function, altogether excluding a THF-dependent enzymatic role. This conclusion is corroborated by in vitro findings showing that HsIBA57 is fully functional in [4Fe–4S] cluster maturation of aconitase without folate supplementation (
      • Weiler B.D.
      • Bruck M.C.
      • Kothe I.
      • Bill E.
      • Lill R.
      • Muhlenhoff U.
      Mitochondrial [4Fe-4S] protein assembly involves reductive [2Fe-2S] cluster fusion on ISCA1-ISCA2 by electron flow from ferredoxin FDX2.
      ). Collectively, several independent functional and structural approaches suggest that mitochondrial Iba57, unlike its THF-dependent protein relatives, does not make use of folates for its function in mitochondrial [4Fe–4S] protein biogenesis.
      Iba57 and YgfZ perform a shared function in [4Fe–4S] protein maturation as indicated by the complementation of an EcYgfZ deletion mutant by various mitochondrial Iba57 members (
      • Waller J.C.
      • Alvarez S.
      • Naponelli V.
      • Lara-Nunez A.
      • Blaby I.K.
      • Da Silva V.
      • et al.
      A role for tetrahydrofolates in the metabolism of iron-sulfur clusters in all domains of life.
      ,
      • Waller J.C.
      • Ellens K.W.
      • Alvarez S.
      • Loizeau K.
      • Ravanel S.
      • Hanson A.D.
      Mitochondrial and plastidial COG0354 proteins have folate-dependent functions in iron-sulphur cluster metabolism.
      ). Conversely, as shown here, EcYgfZ could not replace Iba57 function in yeast, clearly documenting differences between these proteins. A major difference between Iba57 and EcYgfZ appears to be their respective substrate spectrum. While Iba57 is indispensable for maturation of virtually all mitochondrial [4Fe–4S] proteins in yeast and humans, deletion of ygfZ in E. coli mainly affects the activity of the molybdopterin-containing dimethyl sulfoxide reductase (98% reduction; (
      • Gelling C.
      • Dawes I.W.
      • Richhardt N.
      • Lill R.
      • Muhlenhoff U.
      Mitochondrial Iba57p is required for Fe/S cluster formation on aconitase and activation of radical SAM enzymes.
      ,
      • Sheftel A.D.
      • Wilbrecht C.
      • Stehling O.
      • Niggemeyer B.
      • Elsasser H.P.
      • Muhlenhoff U.
      • et al.
      The human mitochondrial ISCA1, ISCA2, and IBA57 proteins are required for [4Fe-4S] protein maturation.
      ,
      • Waller J.C.
      • Alvarez S.
      • Naponelli V.
      • Lara-Nunez A.
      • Blaby I.K.
      • Da Silva V.
      • et al.
      A role for tetrahydrofolates in the metabolism of iron-sulfur clusters in all domains of life.
      ,
      • Waller J.C.
      • Ellens K.W.
      • Alvarez S.
      • Loizeau K.
      • Ravanel S.
      • Hanson A.D.
      Mitochondrial and plastidial COG0354 proteins have folate-dependent functions in iron-sulphur cluster metabolism.
      ,
      • Waller J.C.
      • Ellens K.W.
      • Hasnain G.
      • Alvarez S.
      • Rocca J.R.
      • Hanson A.D.
      Evidence that the folate-dependent proteins YgfZ and MnmEG have opposing effects on growth and on activity of the iron-sulfur enzyme MiaB.
      ,
      • Yu H.
      • Kim K.S.
      YgfZ contributes to secretion of cytotoxic necrotizing factor 1 into outer-membrane vesicles in Escherichia coli.
      )). Formation of the MiaB product 5-methylaminomethyl-2-thiouridine (mnm5s2U) as well as succinate dehydrogenase and fumarase activities are impaired by only ∼40%, whereas other Fe–S protein activities such as those of aconitase and sulfite reductase are even increased upon ygfZ deletion (
      • Waller J.C.
      • Alvarez S.
      • Naponelli V.
      • Lara-Nunez A.
      • Blaby I.K.
      • Da Silva V.
      • et al.
      A role for tetrahydrofolates in the metabolism of iron-sulfur clusters in all domains of life.
      ). The broad and narrow substrate specificities of Iba57 and EcYgfZ, respectively, are well reflected by the fact that deletion of ygfZ in E. coli is not lethal, unlike that of the ISA1 homolog erpA, encoding the potential EcYgfZ partner protein (
      • Loiseau L.
      • Gerez C.
      • Bekker M.
      • Ollagnier-de Choudens S.
      • Py B.
      • Sanakis Y.
      • et al.
      ErpA, an iron sulfur (Fe S) protein of the A-type essential for respiratory metabolism in Escherichia coli.
      ). A further distinction between Iba57 and EcYgfZ concerns the immediate partner proteins. Mitochondrial Iba57 has gained a binding partner, namely the A-type ISC factor Isa2, that has no known functional counterpart in bacteria (
      • Gelling C.
      • Dawes I.W.
      • Richhardt N.
      • Lill R.
      • Muhlenhoff U.
      Mitochondrial Iba57p is required for Fe/S cluster formation on aconitase and activation of radical SAM enzymes.
      ,
      • Beilschmidt L.K.
      • Ollagnier de Choudens S.
      • Fournier M.
      • Sanakis I.
      • Hograindleur M.A.
      • Clemancey M.
      • et al.
      ISCA1 is essential for mitochondrial Fe4S4 biogenesis in vivo.
      ,
      • Gourdoupis S.
      • Nasta V.
      • Calderone V.
      • Ciofi-Baffoni S.
      • Banci L.
      IBA57 recruits ISCA2 to form a [2Fe-2S] cluster-mediated complex.
      ). None of the three bacterial A-type ISC proteins IscA, SufA, or ErpA can functionally replace Isa2 in yeast, in contrast to Isa1 (
      • Muhlenhoff U.
      • Richter N.
      • Pines O.
      • Pierik A.J.
      • Lill R.
      Specialized function of yeast Isa1 and Isa2 proteins in the maturation of mitochondrial [4Fe-4S] proteins.
      ). Together, these genetic features document the profound phenotypical differences of mitochondrial Iba57 and bacterial YgfZ proteins, despite some basic functional overlap.
      Can a different folate dependence of Iba57 and EcYgfZ proteins provide an explanation for these differences? So far, a folate requirement for Fe–S protein maturation in E. coli has been reported only for MiaB (
      • Waller J.C.
      • Alvarez S.
      • Naponelli V.
      • Lara-Nunez A.
      • Blaby I.K.
      • Da Silva V.
      • et al.
      A role for tetrahydrofolates in the metabolism of iron-sulfur clusters in all domains of life.
      ). E. coli folate mutants showed a ∼30% lower MiaB product formation compared with wildtype bacteria, similar to what was found for a ygfZ deletion mutant (see aforementioned). This rather weak defect indicates that folate is not essential for MiaB Fe-S cluster assembly and/or function. Moreover, our comparative structural analyses of both the putative THF-binding pocket and the lipoyl channel of EcYgfZ compared with those present in HsGcvT identify conspicuous differences. Even though in EcYgfZ, the THF-binding pocket may still be large enough to accommodate a THF molecule and the THF entry tunnel is still partially open, many of the specific THF-coordinating residues within the binding pocket are altered in a way making THF binding unlikely. Moreover, the Asp101 residue that is essential for THF-dependent catalysis in HsGcvT is mutated, and the positively charged surface required for interaction with the THF polyglutamyl tail is virtually missing. The lipoyl tunnel is even completely absent in EcYgfZ clearly excluding a binding of this cofactor. Collectively, the maintenance of a cavity in EcYgfZ is in principle compatible with THF binding, yet the largely altered biochemical environment of the putative THF-binding pocket renders stable THF binding and THF-dependent catalysis as seen in GcvT unlikely. Overall, this is consistent with the observed weak binding of THF to EcYgfZ in the millimolar range in vitro (
      • Teplyakov A.
      • Obmolova G.
      • Sarikaya E.
      • Pullalarevu S.
      • Krajewski W.
      • Galkin A.
      • et al.
      Crystal structure of the YgfZ protein from Escherichia coli suggests a folate-dependent regulatory role in one-carbon metabolism.
      ,
      • Waller J.C.
      • Alvarez S.
      • Naponelli V.
      • Lara-Nunez A.
      • Blaby I.K.
      • Da Silva V.
      • et al.
      A role for tetrahydrofolates in the metabolism of iron-sulfur clusters in all domains of life.
      ) and explains why the cofactor was neither coisolated with the protein nor present in the crystal structures, as found for HsGcvT. These considerations may make it necessary to reinspect the THF requirement of bacterial Fe–S protein biogenesis in general and the impact of THF binding for YgfZ function in particular. Taken together, our structural analyses suggest that mitochondrial Iba57 and likely also bacterial YgfZ do not require THF to execute their partially shared function in [4Fe–4S] cluster formation.

      Experimental procedures

      Strains, growth conditions, and recombinant proteins

      Yeast strains (Table S2) were cultivated in synthetic complete minimal medium supplemented with the required amino acids, 2% (w/v) carbon source and¸ when required, 100 μg/ml folic acid or 100 μg/ml dTMP (
      • Sherman F.
      Getting started with yeast.
      ). Plasmids are compiled in Table S3. For purification of recombinant CtIba57, the open reading frame of CtIba57 (codons 61–476) was fused to an N-terminal HIS tag in vector pRSFduet1. E. coli Bl21(DE3) cells were grown in LB medium at 37 °C to an absorbance of 0.5 at 600 nm. Protein expression was induced with IPTG (1 mM final), and cells were cultivated overnight at 28 °C. CtIBA57 was purified by nickel–nitrilotriacetic acid chromatography followed by gel filtration on a Superdex S200 column in buffer P (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, and 5% glycerol).

      Crystal structure of recombinant CtIba57

      CtIba57 was concentrated to 35 mg/ml and crystallized in 0.1 M citric acid (pH 4.0) and 5% (w/v) PEG 6000 after 40 to 60 days incubation at ambient temperature. Crystals were flash frozen in liquid nitrogen employing a cryosolution that consisted of mother liquor supplemented with 30% (v/v) glycerol. Data were collected under cryogenic conditions at the European Synchrotron Radiation Facility at ID29. The crystal structure of CtIba57 (PDB code: 7Z3H) was solved by molecular replacement using the structure of the HsIBA57 (PDB code: 6QE4) as search model (Table S1, (
      • Gourdoupis S.
      • Nasta V.
      • Calderone V.
      • Ciofi-Baffoni S.
      • Banci L.
      IBA57 recruits ISCA2 to form a [2Fe-2S] cluster-mediated complex.
      )).

      Miscellaneous methods

      Statistical analyses were carried out with GraphPad Prism 3 (GraphPad Software, Inc). Errors bars indicate the SEM. The following published methods were used: manipulation of DNA and PCR (
      • Sambrook J.
      • Russel D.W.
      Molecular Cloning - A Laboratory Manual.
      ), transformation of yeast cells (
      • Gietz R.D.
      • Woods R.A.
      Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method.
      ), preparation of yeast mitochondria and cell extracts (
      • Diekert K.
      • de Kroon A.I.
      • Kispal G.
      • Lill R.
      Isolation and subfractionation of mitochondria from the yeast Saccharomyces cerevisiae.
      ), immunological techniques (
      • Greenfield E.A.
      Antibodies - A Laboratory Manual.
      ), and determination of enzyme activities in cell extracts (
      • Molik S.
      • Lill R.
      • Muhlenhoff U.
      Methods for studying iron metabolism in yeast mitochondria.
      ).

      Data availability

      All data are contained within the article and the accompanying supporting information.

      Supporting information

      This article contains supporting information (
      • Weiler B.D.
      • Bruck M.C.
      • Kothe I.
      • Bill E.
      • Lill R.
      • Muhlenhoff U.
      Mitochondrial [4Fe-4S] protein assembly involves reductive [2Fe-2S] cluster fusion on ISCA1-ISCA2 by electron flow from ferredoxin FDX2.
      ,
      • Gelling C.
      • Dawes I.W.
      • Richhardt N.
      • Lill R.
      • Muhlenhoff U.
      Mitochondrial Iba57p is required for Fe/S cluster formation on aconitase and activation of radical SAM enzymes.
      ,
      • Muhlenhoff U.
      • Richter N.
      • Pines O.
      • Pierik A.J.
      • Lill R.
      Specialized function of yeast Isa1 and Isa2 proteins in the maturation of mitochondrial [4Fe-4S] proteins.
      ,
      • Okamura-Ikeda K.
      • Hosaka H.
      • Yoshimura M.
      • Yamashita E.
      • Toma S.
      • Nakagawa A.
      • et al.
      Crystal structure of human T-protein of glycine cleavage system at 2.0 A resolution and its implication for understanding non-ketotic hyperglycinemia.
      ,
      • Gietz R.D.
      • Woods R.A.
      Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method.
      ,
      • Mortimer R.K.
      • Johnston J.R.
      Genealogy of principal strains of the yeast genetic stock center.
      ,
      • Brachmann C.B.
      • Davies A.
      • Cost G.J.
      • Caputo E.
      • Li J.
      • Hieter P.
      • et al.
      Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications.
      ,
      • Janke C.
      • Magiera M.M.
      • Rathfelder N.
      • Taxis C.
      • Reber S.
      • Maekawa H.
      • et al.
      A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes.
      ,
      • Gueldener U.
      • Heinisch J.
      • Koehler G.J.
      • Voss D.
      • Hegemann J.H.
      A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast.
      ,
      • Muhlenhoff U.
      • Richhardt N.
      • Ristow M.
      • Kispal G.
      • Lill R.
      The yeast frataxin homolog Yfh1p plays a specific role in the maturation of cellular Fe/S proteins.
      ,
      • Funk M.
      • Niedenthal R.
      • Mumberg D.
      • Brinkmann K.
      • Ronicke V.
      • Henkel T.
      Vector systems for heterologous expression of proteins in Saccharomyces cerevisiae.
      ).

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgments

      We thank Devid Mrusek for help during crystallization of CtIBA57. We acknowledge the contribution of the Core Facility “Protein Biochemistry and Spectroscopy” of Philipps-Universität Marburg.

      Author contributions

      U. M. conceptualization; U. M. methodology; U. M., B. D. W., F. N., R. M., I. K., and F. A. investigation; U. M. writing–original draft; R. L. writing–review & editing; S.-A. F. and F. A. visualization; G. B. and R. L. supervision; R. L. project administration; R. L. funding acquisition.

      Funding and additional information

      The work was financially supported by Deutsche Forschungsgemeinschaft through funds from SFB 987 (to R.L. and U.M.) and SPPs 1710 and 1927 (to R.L.).

      Supporting information

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