NifS-mediated assembly of [4Fe-4S] clusters in the N- and C-terminal domains of the NifU scaffold protein

NifU is a homodimeric modular protein comprising N- and C-terminal domains and a central domain with a redox-active [2Fe-2S](2+,+) cluster. It plays a crucial role as a scaffold protein for the assembly of the Fe-S clusters required for the maturation of nif-specific Fe-S proteins. In this work, the time course and products of in vitro NifS-mediated iron-sulfur cluster assembly on full-length NifU and truncated forms involving only the N-terminal domain or the central and C-terminal domains have been investigated using UV-vis absorption and M?ssbauer spectroscopies, coupled with analytical studies. The results demonstrate sequential assembly of labile [2Fe-2S](2+) and [4Fe-4S](2+) clusters in the U-type N-terminal scaffolding domain and the assembly of [4Fe-4S](2+) clusters in the Nfu-type C-terminal scaffolding domain. Both scaffolding domains of NifU are shown to be competent for in vitro maturation of nitrogenase component proteins, as evidenced by rapid transfer of [4Fe-4S](2+) clusters preassembled on either the N- or C-terminal domains to the apo nitrogenase Fe protein. Mutagenesis studies indicate that a conserved aspartate (Asp37) plays a critical role in mediating cluster transfer. The assembly and transfer of clusters on NifU are compared with results reported for U- and Nfu-type scaffold proteins, and the need for two functional Fe-S cluster scaffolding domains on NifU is discussed.     

A proposed role for the Azotobacter vinelandii NfuA protein as an intermediate iron-sulfur cluster carrier

Iron-sulfur clusters ([Fe-S] clusters) are assembled on molecular scaffolds and subsequently used for maturation of proteins that require [Fe-S] clusters for their functions. Previous studies have shown that Azotobacter vinelandii produces at least two [Fe-S] cluster assembly scaffolds: NifU, required for the maturation of nitrogenase, and IscU, required for the general maturation of other [Fe-S] proteins. A. vinelandii also encodes a protein designated NfuA, which shares amino acid sequence similarity with the C-terminal region of NifU. The activity of aconitase, a [4Fe-4S] cluster-containing enzyme, is markedly diminished in a strain containing an inactivated nfuA gene. This inactivation also results in a null-growth phenotype when the strain is cultivated under elevated oxygen concentrations. NifU has a limited ability to serve the function of NfuA, as its expression at high levels corrects the defect of the nfuA-disrupted strain. Spectroscopic and analytical studies indicate that one [4Fe-4S] cluster can be assembled in vitro within a dimeric form of NfuA. The resultant [4Fe-4S] cluster-loaded form of NfuA is competent for rapid in vitro activation of apo-aconitase. Based on these results a model is proposed where NfuA could represent a class of intermediate [Fe-S] cluster carriers involved in [Fe-S] protein maturation.     

Modular organization and identification of a mononuclear iron-binding site within the NifU protein

The NifS and NifU nitrogen fixation-specific gene products are required for the full activation of both the Fe-protein and MoFe-protein of nitrogenase from Azotobacter vinelandii. Because the two nitrogenase component proteins both require the assembly of [Fe-S]-containing clusters for their activation, it has been suggested that NifS and NifU could have complementary functions in the mobilization of sulfur and iron necessary for nitrogenase-specific [Fe-S] cluster assembly. The NifS protein has been shown to have cysteine desulfurase activity and can be used to supply sulfide for the in vitro catalytic formation of [Fe-S] clusters. The NifU protein was previously purified and shown to be a homodimer with a [2Fe-2S] cluster in each subunit. In the present work, primary sequence comparisons, amino acid substitution experiments, and optical and resonance Raman spectroscopic characterization of recombinantly produced NifU and NifU fragments are used to show that NifU has a modular structure. One module is contained in approximately the N-terminal third of NifU and is shown to provide a labile rubredoxin-like ferric-binding site. Cysteine residues Cys³⁵, Cys⁶², and Cys¹⁰⁶ are necessary for binding iron in the rubredoxin-like mode and visible extinction coefficients indicate that up to one ferric ion can be bound per NifU monomer. The second module is contained in approximately the C-terminal half of NifU and provides the [2Fe-2S] cluster-binding site. Cysteine residues Cys¹³⁷, Cys¹³⁹, Cys¹⁷², and Cys¹⁷⁵ provide ligands to the [2Fe-2S] cluster. The cysteines involved in ligating the mononuclear Fe in the rubredoxin-like site and those that provide the [2Fe-2S] cluster ligands are all required for the full physiological function of NifU. The only two other cysteines contained within NifU, Cys²⁷² and Cys²⁷⁵, are not necessary for iron binding at either site, nor are they required for the full physiological function of NifU. The results provide the basis for a model where iron bound in labile rubredoxin-like sites within NifU is used for [Fe-S] cluster formation. The [2Fe-2S] clusters contained within NifU are proposed to have a redox function involving the release of Fe from bacterioferritin and/or the release of Fe or an [Fe-S] cluster precursor from the rubredoxin-like binding site. Received: 27 October 1999 / Accepted: 30 November 1999     

Characterization of the NifU and NifS Fe-S cluster formation proteins essential for viability in Helicobacter pylori

The Fe-S cluster formation proteins NifU and NifS are essential for viability in the ulcer causing human pathogen Helicobacter pylori. Obtaining viable H. pylori mutants upon mutagenesis of the genes encoding NifU and NifS was unsuccessful even by growing the potential transformants under many different conditions including low O(2) atmosphere and supplementation with both ferric and ferrous iron. When a second copy of nifU was introduced into the chromosome at a unrelated site, creating a mero-diploid strain for nifU, this second copy of the gene could be disrupted at high frequency. This indicates that the procedures used for transformation were capable of nifU mutagenesis, so that the failure to recover mutants is solely due to the requirement of nifU for H. pylori viability. H. pylori NifU and NifS were expressed in Escherichia coli and purified to near homogeneity, and the proteins were characterized. Purified NifU is a red protein that contains approximately 1.5 atoms of iron per monomer. This iron was determined to be in the form of a redox-active [2Fe-2S](2+,+) cluster by characteristic UV-visible, EPR, and MCD spectra. The primary structure of NifU also contains the three conserved cysteine residues which are involved in providing the scaffold for the assembly of a transient Fe-S cluster for insertion into apoprotein. Purified NifS has a yellow color and UV-visible spectra characteristic of a pyridoxal phosphate containing enzyme. NifS is a cysteine desulfurase, releasing sulfur or sulfide (depending on the reducing environment) from L-cysteine, in agreement with its proposed role as a sulfur donor to Fe-S clusters. The results here indicate that the NifU type of Fe-S cluster formation proteins is not specific for maturation of the nitrogenase proteins and, as H. pylori lacks other Fe-S cluster assembly proteins, that the H. pylori NifS and NifU are responsible for the assembly of many (non-nitrogenase) Fe-S clusters.     

Controlled expression of nif and isc iron-sulfur protein maturation components reveals target specificity and limited functional replacement between the two systems

The nitrogen-fixing organism Azotobacter vinelandii contains at least two systems that catalyze formation of [Fe-S] clusters. One of these systems is encoded by nif genes, whose products supply [Fe-S] clusters required for maturation of nitrogenase. The other system is encoded by isc genes, whose products are required for maturation of [Fe-S] proteins that participate in general metabolic processes. The two systems are similar in that they include an enzyme for the mobilization of sulfur (NifS or IscS) and an assembly scaffold (NifU or IscU) upon which [Fe-S] clusters are formed. Normal cellular levels of the Nif system, which supplies [Fe-S] clusters for the maturation of nitrogenase, cannot also supply [Fe-S] clusters for the maturation of other cellular [Fe-S] proteins. Conversely, when produced at the normal physiological levels, the Isc system cannot supply [Fe-S] clusters for the maturation of nitrogenase. In the present work we found that such target specificity for IscU can be overcome by elevated production of NifU. We also found that NifU, when expressed at normal levels, is able to partially replace the function of IscU if cells are cultured under low-oxygen-availability conditions. In contrast to the situation with IscU, we could not establish conditions in which the function of IscS could be replaced by NifS. We also found that elevated expression of the Isc components, as a result of deletion of the regulatory iscR gene, improved the capacity for nitrogen-fixing growth of strains deficient in either NifU or NifS.     

IscA, an alternate scaffold for Fe-S cluster biosynthesis.

An IscA homologue within the nif regulon of Azotobacter vinelandii, designated (Nif)IscA, was expressed in Escherichia coli and purified to homogeneity. Purified (Nif)IscA was found to be a homodimer of 11-kDa subunits that contained no metal centers or other prosthetic groups in its as-isolated form. Possible roles for (Nif)IscA in Fe-S cluster biosynthesis were assessed by investigating the ability to bind iron and to assemble Fe-S clusters in a NifS-directed process, as monitored by the combination of UV-vis absorption, M?ssbauer, resonance Raman, variable-temperature magnetic circular dichroism, and EPR spectroscopies. Although (Nif)IscA was found to bind ferrous ion in a tetrahedral, predominantly cysteinyl-ligated coordination environment, the low-binding affinity argues against a specific role as a metallochaperone for the delivery of ferrous ion to other Fe-S cluster assembly proteins. Rather, a role for (Nif)IscA as an alternate scaffold protein for Fe-S cluster biosynthesis is proposed, based on the NifS-directed assembly of approximately one labile [4Fe-4S](2+) cluster per (Nif)IscA homodimer, via a transient [2Fe-2S](2+) cluster intermediate. The cluster assembly process was monitored temporally using UV-vis absorption and M?ssbauer spectroscopy, and the intermediate [2Fe-2S](2+)-containing species was additionally characterized by resonance Raman spectroscopy. The M?ssbauer and resonance Raman properties of the [2Fe-2S](2+) center are consistent with complete cysteinyl ligation. The presence of three conserved cysteine residues in all IscA proteins and the observed cluster stoichiometry of approximately one [2Fe-2S](2+) or one [4Fe-4S](2+) per homodimer suggest that both cluster types are subunit bridging. In addition, (Nif)IscA was shown to couple delivery of iron and sulfur by using ferrous ion to reduce sulfane sulfur. The ability of Fe-S scaffold proteins to couple the delivery of these two toxic and reactive Fe-S cluster precursors is likely to be important for minimizing the cellular concentrations of free ferrous and sulfide ions. On the basis of the spectroscopic and analytical results, mechanistic schemes for NifS-directed cluster assembly on (Nif)IscA are proposed. It is proposed that the IscA family of proteins provide alternative scaffolds to the NifU and IscU proteins for mediating nif-specific and general Fe-S cluster assembly.     

The NMR structure of the domain II of a chloroplastic NifU-like protein OsNifU1A

NifU-like proteins are a highly conserved protein that serves as the scaffold for assembly of Fe-S clusters. Chloroplastic NifU-like proteins have tandem NifU like domains, named domain I and domain II. Although the amino acid sequences of these domains are very similar to each other, the predicted functional region for the Fe-S cluster assembly, the CXXC motif, exists only in domain I. The structure of the domain II of chloroplastic NifU-like protein OsNifU1A has an α-β sandwich structure containing two α helices located on one side of the β-sheet. The electrostatic surface potential of OsNifU1A domain II is predominantly positively charged. Chloroplastic NifU-like proteins are targeted to ferredoxin for transferring the Fe-S cluster. The ferredoxin presents an overall negatively charged surface, which may evoke an electrostatic association with OsNifU1A domain II.     

Solution NMR structure of the iron-sulfur cluster assembly protein U (IscU) with zinc bound at the active site

IscU is a highly conserved protein that serves as the scaffold for IscS-mediated assembly of iron-sulfur ([Fe-S]) clusters. We report the NMR solution structure of monomeric Haemophilus influenzae IscU with zinc bound at the [Fe-S] cluster assembly site. The compact core of the globular structure has an alpha-beta sandwich architecture with a three-stranded antiparallel beta-sheet and four alpha-helices. A nascent helix is located N-terminal to the core structure. The zinc is ligated by three cysteine residues and one histidine residue that are located in and near conformationally dynamic loops at one end of the IscU structure. Removal of the zinc metal by chelation results in widespread loss of structure in the apo form. The zinc-bound IscU may be a good model for iron-loaded IscU and may demonstrate structural features found in the [Fe-S] cluster bound form. Structural and functional similarities, genomic context in operons containing other homologous genes, and distributions of conserved surface residues support the hypothesis that IscU protein domains are homologous (i.e. derived from a common ancestor) with the SufE/YgdK family of [Fe-S] cluster assembly proteins.     

The primary structure of a protein containing a putative [6Fe-6S] prismane cluster from Desulfovibrio vulgaris (Hildenborough).

The gene encoding a protein containing a putative [6Fe-6S] prismane cluster has been cloned from Desulfovibrio vulgaris (Hildenborough) and sequenced. The gene encodes a polypeptide composed of 553 amino acids (60,161 Da). The DNA-derived amino acid sequence was partly confirmed by N-terminal sequencing of the purified protein and of fragments of the protein generated by CNBr cleavage. Furthermore, the C-terminal sequence was verified by digestion with carboxypeptidases A and B. The polypeptide contains nine Cys residues. Four of these residues are gathered in a Cys-Xaa2-Cys-Xaa7-Cys-Xaa5-Cys motif located towards the N-terminus of the protein. No relevant sequence similarity was found with other proteins, including those with high-spin Fe-S clusters (nitrogenase, hydrogenase), with one significant exception: the stretch containing the first four Cys residues spans two submotifs, Cys-Xaa2-Cys and Lys-Gly-Xaa-Cys-Gly, separated by 11 residues, that are also present in high-spin Fe-S cluster containing CO dehydrogenase. Western-blot analysis demonstrates cross-reactivity of antibodies raised against the purified protein both in Desulfovibrio strains and other sulfate-reducing bacteria. Hybridization of the cloned gene with genomic DNA of several other Desulfovibrio species indicates that homologous sequences are generally present in the genus Desulfovibrio.     

Evidence for nifU and nifS participation in the biosynthesis of the iron-molybdenum cofactor of nitrogenase

The nifU and nifS genes encode the components of a cellular machinery dedicated to the assembly of [2Fe-2S] and [4Fe-4S] clusters required for growth under nitrogen-fixing conditions. The NifU and NifS proteins are involved in the production of active forms of the nitrogenase component proteins, NifH and NifDK. Although NifH contains a [4Fe-4S] cluster, the NifDK component carries two complex metalloclusters, the iron-molybdenum cofactor (FeMo-co) and the [8Fe-7S] P-cluster. FeMo-co, located at the active site of NifDK, is composed of 7 iron, 9 sulfur, 1 molybdenum, 1 homocitrate, and 1 unidentified light atom. To investigate whether NifUS are required for FeMo-co biosynthesis and to understand at what level(s) they might participate in this process, we analyzed the effect of nifU and nifS mutations on the formation of active NifB protein and on the accumulation of NifB-co, an isolatable intermediate of the FeMo-co biosynthetic pathway synthesized by the product of the nifB gene. The nifU and nifS genes were required to accumulate NifB-co in a nifN mutant background. This result clearly demonstrates the participation of NifUS in NifB-co synthesis and suggests a specific role of NifUS as the major provider of [Fe-S] clusters that serve as metabolic substrates for the biosynthesis of FeMo-co. Surprisingly, although nifB expression was attenuated in nifUS mutants, the assembly of the [Fe-S] clusters of NifB was compensated by other non-nif machinery for the assembly of [Fe-S] clusters, indicating that NifUS are not essential to synthesize active NifB.     

The crystal structure of DR2241 from Deinococcus radiodurans at 1.9 A resolution reveals a multi-domain protein with structural similarity to chelatases but also with two additional novel domains

A unique family of proteins have been identified in the Deinococcus genus with an N-terminal cobalamin (vitamin B(12)) chelatase domain denoted CbiX and an additional unique C-terminal domain with unknown function. Here we report the first crystal structure from this new family of proteins with the structure of Deinococcus radiodurans protein DR2241. The structure reveals a multi-domain protein where domains A (residues 1-132) has the same fold as the small CbiX (CbiX(S)), domains A and B (residues 1-272) follow the chelatase super-family fold and the two additional unique domains C and D have no structural homologues. Domain D harbours the sequence motifs CxxC and CxxxC, in which DR2241 gives the first evidence that these motifs bind a [4Fe-4S] iron-sulphur cluster. In solution there are indications of multimeric forms, and in the crystallographic asymmetric unit a tetramer is found where domains C and D are involved in stabilising the tetrameric assembly.     

The UDP-glucose:glycoprotein glucosyltransferase is organized in at least two tightly bound domains from yeast to mammals

The endoplasmic reticulum UDP-Glc:glycoprotein glucosyltransferase (GT) exclusively glucosylates nonnative glycoprotein conformers. GT sequence analysis suggests that it is composed of at least two domains: the N-terminal domain, which composes 80% of the molecule, has no significant similarity to other known proteins and was proposed to be involved in the recognition of non-native conformers and the C-terminal or catalytic domain, which displays a similar size and significant similarity to members of glycosyltransferase family 8. Here, we show that N- and C-terminal domains from Rattus norvegicus and Schizosaccharomyces pombe GTs remained tightly but not covalently bound upon a mild proteolytic treatment and could not be separated without loss of enzymatic activity. The notion of a two-domain protein was reinforced by the synthesis of an active enzyme upon transfection of S. pombe GT null mutants with two expression vectors, each of them encoding one of both domains. Transfection with the C-terminal domain-encoding vector alone yielded an inactive, rapidly degraded protein, thus indicating that the N-terminal domain is required for proper folding of the C-terminal catalytic portion. If, indeed, the N-terminal domain is, as proposed, also involved in glycoprotein conformation recognition, the tight association between N- and C-terminal domains may explain why only N-glycans in close proximity to protein structural perturbations are glucosylated by the enzyme. Although S. pombe and Drosophila melanogaster GT N-terminal domains display an extremely poor similarity (16.3%), chimeras containing either yeast N-terminal and fly C-terminal domains or the inverse construction were enzymatically and functionally active in vivo, thus indicating that the N-terminal domains of both GTs shared three-dimensional features.     

Biosynthesis of iron-sulphur clusters is a complex and highly conserved process

Iron-sulphur ([Fe-S]) clusters are simple inorganic prosthetic groups that are contained in a variety of proteins having functions related to electron transfer, gene regulation, environmental sensing and substrate activation. In spite of their simple structures, biological [Fe-S] clusters are not formed spontaneously. Rather, a consortium of highly conserved proteins is required for both the formation of [Fe-S] clusters and their insertion into various protein partners. Among the [Fe-S] cluster biosynthetic proteins are included a pyridoxal phosphate-dependent enzyme (NifS) that is involved in the activation of sulphur from l-cysteine, and a molecular scaffold protein (NifU) upon which [Fe-S] cluster precursors are formed. The formation or transfer of [Fe-S] clusters appears to require an electron-transfer step. Another complexity is that molecular chaperones homologous to DnaJ and DnaK are involved in some aspect of the maturation of [Fe-S]-cluster-containing proteins. It appears that the basic biochemical features of [Fe-S] cluster formation are strongly conserved in Nature, since organisms from all three life Kingdoms contain the same consortium of homologous proteins required for [Fe-S] cluster formation that were discovered in the eubacteria.     

A vertebrate-type ferredoxin domain in the Na+-translocating NADH dehydrogenase from Vibrio cholerae

The Na(+)-translocating NADH:quinone oxidoreductase from Vibrio cholerae contains a single Fe-S cluster localized in subunit NqrF. Here we study the electronic properties of the Fe-S center in a truncated version of the NqrF subunit comprising only its ferredoxin-like Fe-S domain. M?ssbauer spectroscopy of the Fe-S domain in the oxidized state is consistent with a binuclear Fe-S cluster with tetrahedral sulfur coordination by the cysteine residues Cys(70), Cys(76), Cys(79), and Cys(111). Important sequence motifs surrounding these cysteines are conserved in the Fe-S domain and in vertebrate-type ferredoxins. The magnetic circular dichroism spectra of the photochemically reduced Fe-S domain exhibit a striking similarity to the magnetic circular dichroism spectra of vertebrate-type ferredoxins required for the in vivo assembly of iron-sulfur clusters. This study reveals a novel function for vertebrate-type [2Fe-2S] clusters as redox cofactors in respiratory dehydrogenases.     

A bridging [4Fe-4S] cluster and nucleotide binding are essential for function of the Cfd1-Nbp35 complex as a scaffold in iron-sulfur protein maturation

The essential P-loop NTPases Cfd1 and Nbp35 of the cytosolic iron-sulfur (Fe-S) protein assembly machinery perform a scaffold function for Fe-S cluster synthesis. Both proteins contain a nucleotide binding motif of unknown function and a C-terminal motif with four conserved cysteine residues. The latter motif defines the Mrp/Nbp35 subclass of P-loop NTPases and is suspected to be involved in transient Fe-S cluster binding. To elucidate the function of these two motifs, we first created cysteine mutant proteins of Cfd1 and Nbp35 and investigated the consequences of these mutations by genetic, cell biological, biochemical, and spectroscopic approaches. The two central cysteine residues (CPXC) of the C-terminal motif were found to be crucial for cell viability, protein function, coordination of a labile [4Fe-4S] cluster, and Cfd1-Nbp35 hetero-tetramer formation. Surprisingly, the two proximal cysteine residues were dispensable for all these functions, despite their strict evolutionary conservation. Several lines of evidence suggest that the C-terminal CPXC motifs of Cfd1-Nbp35 coordinate a bridging [4Fe-4S] cluster. Upon mutation of the nucleotide binding motifs Fe-S clusters could no longer be assembled on these proteins unless wild-type copies of Cfd1 and Nbp35 were present in trans. This result indicated that Fe-S cluster loading on these scaffold proteins is a nucleotide-dependent step. We propose that the bridging coordination of the C-terminal Fe-S cluster may be ideal for its facile assembly, labile binding, and efficient transfer to target Fe-S apoproteins, a step facilitated by the cytosolic iron-sulfur (Fe-S) protein assembly proteins Nar1 and Cia1 in vivo.     

Purification of a NifEN protein complex that contains bound molybdenum and a FeMo-Co precursor from an Azotobacter vinelandii DeltanifHDK strain

The NifEN protein complex serves as a molecular scaffold where some of the steps for the assembly of the iron-molybdenum cofactor (FeMo-co) of nitrogenase take place. A His-tagged version of the NifEN complex has been previously purified and shown to carry two identical [4Fe-4S] clusters of unknown function and a [Fe-S]-containing FeMo-co precursor. We have improved the purification of the his-NifEN protein from a DeltanifHDK strain of Azotobacter vinelandii and have found that the amounts of iron and molybdenum within NifEN were significantly higher than those reported previously. In an in vitro FeMo-co synthesis system with purified components, the NifEN protein served as a source of both molybdenum and a [Fe-S]-containing FeMo-co precursor, showing significant FeMo-co synthesis activity in the absence of externally added molybdate. Thus, the NifEN scaffold protein, purified from DeltanifHDK background, contained the Nif-Bco-derived Fe-S cluster and molybdenum, although these FeMo-co constituents were present at different levels within the protein complex.     

The structure of aconitase

The crystal structure of the 80,000 Da Fe-S enzyme aconitase has been solved and refined at 2.1 A resolution. The protein contains four domains; the first three from the N-terminus are closely associated around the [3Fe-4S] cluster with all three cysteine ligands to the cluster being provided by the third domain. Association of the larger C-terminal domain with the first three domains creates an extensive cleft leading to the Fe-S cluster. Residues from all four domains contribute to the active site region, which is defined by the Fe-S cluster and a bound SO4(2-) ion. This region of the structure contains 4 Arg, 3 His, 3 Ser, 2 Asp, 1 Glu, 3 Asn, and 1 Gln residues, as well as several bound water molecules. Three of these side chains reside on a three-turn 3(10) helix in the first domain. The SO4(2-) ion is bound 9.3 A from the center of the [3Fe-4S] cluster by the side chains of 2 Arg and 1 Gln residues. Each of 3 His side chains in the putative active site is paired with Asp or Glu side chains.     

Shared Active Site Architecture between the Large Subunit of Eukaryotic Primase and DNA Photolyase

Background

DNA synthesis during replication relies on RNA primers synthesised by the primase, a specialised DNA-dependent RNA polymerase that can initiate nucleic acid synthesis de novo. In archaeal and eukaryotic organisms, the primase is a heterodimeric enzyme resulting from the constitutive association of a small (PriS) and large (PriL) subunit. The ability of the primase to initiate synthesis of an RNA primer depends on a conserved Fe-S domain at the C-terminus of PriL (PriL-CTD). However, the critical role of the PriL-CTD in the catalytic mechanism of initiation is not understood.

Methodology/Principal Findings

Here we report the crystal structure of the yeast PriL-CTD at 1.55 Å resolution. The structure reveals that the PriL-CTD folds in two largely independent alpha-helical domains joined at their interface by a [4Fe-4S] cluster. The larger N-terminal domain represents the most conserved portion of the PriL-CTD, whereas the smaller C-terminal domain is largely absent in archaeal PriL. Unexpectedly, the N-terminal domain reveals a striking structural similarity with the active site region of the DNA photolyase/cryptochrome family of flavoproteins. The region of similarity includes PriL-CTD residues that are known to be essential for initiation of RNA primer synthesis by the primase.

Conclusion/Significance

Our study reports the first crystallographic model of the conserved Fe-S domain of the archaeal/eukaryotic primase. The structural comparison with a cryptochrome protein bound to flavin adenine dinucleotide and single-stranded DNA provides important insight into the mechanism of RNA primer synthesis by the primase.     

The Cfd1-Nbp35 complex acts as a scaffold for iron-sulfur protein assembly in the yeast cytosol

Biogenesis of iron-sulfur ([Fe-S]) proteins in eukaryotes requires the function of complex proteinaceous machineries in both mitochondria and cytosol. In contrast to the mitochondrial pathway, little is known about [Fe-S] protein assembly in the cytosol. So far, four highly conserved proteins (Cfd1, Nbp35, Nar1 and Cia1) have been identified as members of the cytosolic [Fe-S] protein assembly machinery, but their molecular function is unresolved. Using in vivo and in vitro approaches, we found that the soluble P-loop NTPases Cfd1 and Nbp35 form a complex and bind up to three [4Fe-4S] clusters, one at the N terminus of Nbp35 and one each at a new C-terminal cysteine-rich motif present in both proteins. These labile [Fe-S] clusters can be rapidly transferred and incorporated into target [Fe-S] apoproteins in a Nar1- and Cia1-dependent fashion. Our data suggest that the Cfd1-Nbp35 complex functions as a novel scaffold for [Fe-S] cluster assembly in the eukaryotic cytosol.