Structural and biochemical investigations of a HEAT-repeat protein involved in the cytosolic iron-sulfur cluster assembly pathway.

Iron-sulfur clusters are essential for life and defects in their biosynthesis lead to human diseases. The mechanism of cluster assembly and delivery to cytosolic and nuclear client proteins via the cytosolic iron-sulfur cluster assembly (CIA) pathway is not well understood. Here we report cryo-EM structures of the HEAT-repeat protein Met18 from Saccharomyces cerevisiae, a key component of the CIA targeting complex (CTC) that identifies cytosolic and nuclear client proteins and delivers a mature iron-sulfur cluster. We find that in the absence of other CTC proteins, Met18 adopts tetrameric and hexameric states. Using mass photometry and negative stain EM, we show that upon the addition of Cia2, these higher order oligomeric states of Met18 disassemble. We also use pulldown assays to identify residues of critical importance for Cia2 binding and recognition of the Leu1 client, many of which are buried when Met18 oligomerizes. Our structures show conformations of Met18 that have not been previously observed in any Met18 homolog, lending support to the idea that a highly flexible Met18 may be key to how the CTC is able to deliver iron-sulfur clusters to client proteins of various sizes and shapes, i.e. Met18 conforms to the dimensions needed.

Cytosolic iron-sulfur protein assembly system identifies clients by a C-terminal tripeptide.

The eukaryotic cytosolic Fe-S protein assembly (CIA) machinery inserts iron-sulfur (Fe-S) clusters into cytosolic and nuclear proteins. In the final maturation step, the Fe-S cluster is transferred to the apo-proteins by the CIA-targeting complex (CTC). However, the molecular recognition determinants of client proteins are unknown. We show that a conserved [LIM]-[DES]-[WF]-COO- tripeptide is present at the C-terminus of more than a quarter of clients or their adaptors. When present, this targeting complex recognition (TCR) motif is necessary and sufficient for binding to the CTC in vitro and for directing Fe-S cluster delivery in vivo. Remarkably, fusion of this TCR signal enables engineering of cluster maturation on a nonnative protein via recruitment of the CIA machinery. Our study advances our understanding of Fe-S protein maturation and paves the way for bioengineering novel pathways containing Fe-S enzymes.

Cytosolic iron-sulfur protein assembly system identifies clients by a C-terminal tripeptide.

The eukaryotic cytosolic Fe-S protein assembly (CIA) machinery inserts iron-sulfur (Fe-S) clusters into cytosolic and nuclear proteins. In the final maturation step, the Fe-S cluster is transferred to the apo-proteins by the CIA-targeting complex (CTC). However, the molecular recognition determinants of client proteins are unknown. We show that a conserved [LIM]-[DES]-[WF]-COO- tripeptide present at the C-terminus of clients is necessary and sufficient for binding to the CTC in vitro and directing Fe-S cluster delivery in vivo. Remarkably, fusion of this TCR (target complex recognition) signal enables engineering of cluster maturation on a non-native protein via recruitment of the CIA machinery. Our study significantly advances our understanding of Fe-S protein maturation and paves the way for bioengineering applications.

Methods to Unravel the Roles of ATPases in Fe-S Cluster Biosynthesis.

Complex biosynthetic pathways are required for the assembly and insertion of iron-sulfur (Fe-S) cluster cofactors. Each of the four cluster biogenesis systems that have been discovered requires at least one ATPase. Generally, the function of nucleotide hydrolysis in Fe-S cluster biogenesis is understudied. For example, the cytosolic Fe-S cluster assembly (CIA) pathway is proposed to begin with a scaffold, which assembles nascent Fe-S clusters destined for cytosolic and nuclear enzymes. This scaffold, comprised of Nbp35 and Cfd1 in yeast, possesses an ATPase site that is necessary for CIA function, but the role of nucleotide hydrolysis is poorly understood. Herein, we describe the in vitro methods that have been developed to uncover how the ATPase site of the scaffold regulates interaction with one of its partner proteins, Dre2. We describe a qualitative affinity copurification assay and a quantitative assay for evaluating the dissociation constant for the scaffold-partner protein complex. Finally, we describe kinetic methods to measure the kcat and KM values for ATP hydrolysis by the scaffold-partner protein complex and the execution of the ATPase assays in an anaerobic environment. These methods could be applied to study other ATPases to advance our mechanistic understanding of nucleotide hydrolases involved in metallocluster biogenesis.

Coupling Nucleotide Binding and Hydrolysis to Iron-Sulfur Cluster Acquisition and Transfer Revealed through Genetic Dissection of the Nbp35 ATPase Site.

The cytosolic iron-sulfur cluster assembly (CIA) scaffold, comprising Nbp35 and Cfd1 in yeast, assembles iron-sulfur (FeS) clusters destined for cytosolic and nuclear enzymes. ATP hydrolysis by the CIA scaffold plays an essential but poorly understood role in cluster biogenesis. Here we find that mutation of conserved residues in the four motifs comprising the ATPase site of Nbp35 diminished the scaffold's ability to both assemble and transfer its FeS cluster in vivo. The mutants fall into four phenotypic classes that can be understood by how each set of mutations affects ATP binding and hydrolysis. In vitro studies additionally revealed that occupancy of the bridging FeS cluster binding site decreases the scaffold's affinity for the nucleotide. On the basis of our findings, we propose that nucleotide binding and hydrolysis by the CIA scaffold drive a series of protein conformational changes that regulate association with other proteins in the pathway and with its newly formed FeS cluster. Our results provide insight into how the ATPase and cluster scaffolding activities are allosterically integrated.

The Cfd1 Subunit of the Nbp35-Cfd1 Iron Sulfur Cluster Scaffolding Complex Controls Nucleotide Binding.

The cytosolic iron sulfur cluster assembly (CIA) scaffold biosynthesizes iron sulfur cluster cofactors for enzymes residing in the cytosol and the nucleus. In fungi and animals, it comprises two homologous ATPases, called Nbp35 and Cfd1 in yeast, which can form homodimeric and heterodimeric complexes. Both proteins are required for CIA function, but their individual roles are not well understood. Here we investigate the nucleotide affinity of each form of the scaffold for ATP and ADP to reveal any differences that could shed light on the functions of the different oligomeric forms of the protein or any distinct roles of the individual subunits. All forms of the CIA scaffold are specific for adenosine nucleotides and not guanosine nucleotides. Although the Cfd1 homodimer has no detectable ATPase activity, it binds ATP with an affinity comparable to that of the hydrolysis competent forms, Nbp352 and Nbp35-Cfd1. Titrations to determine the number of nucleotide binding sites combined with site-directed mutagenesis demonstrate that the nucleotide must bind to the Cfd1 subunit of the heterodimer before it can bind to Nbp35 and that the Cfd1 subunit is hydrolysis competent when bound to Nbp35 in the heterodimer. Altogether, our work reveals the distinct roles of the Nbp35 and Cfd1 subunits in their heterodimeric complex. Cfd1 controls nucleotide binding, and the Nbp35 subunit is required to activate nucleotide hydrolysis.

Approaches to Interrogate the Role of Nucleotide Hydrolysis by Metal Trafficking NTPases: The Nbp35-Cfd1 Iron-Sulfur Cluster Scaffold as a Case Study.

Nucleotide hydrolases play integral yet poorly understood roles in several metallocluster biosynthetic pathways. For example, the cytosolic iron-sulfur cluster assembly (CIA) is initiated by the CIA scaffold, an ATPase which builds new iron-sulfur clusters for proteins localized to the cytosol and the nucleus in eukaryotic organisms. While in vivo studies have demonstrated the scaffold's nucleotide hydrolase domain is vital for its function, in vitro approaches have not revealed tight allosteric coupling between the cluster scaffolding site and the ATPase site. Thus, the role of ATP hydrolysis has been hard to pinpoint. Herein, we describe methods to probe the nucleotide affinity and hydrolysis activity of the CIA scaffold from yeast, which is comprised of two homologous polypeptides called Nbp35 and Cfd1. In particular, we report two different equilibrium binding assays that make use of commercially available fluorescent nucleotide analogs. Importantly, these assays can be applied to probe nucleotide affinity of both the apo- and holo-forms of the CIA scaffold. Generally, these fluorescent nucleotide analogs have been underutilized to probe metal trafficking NTPase because one of the most commonly used probes, mantATP, which is labeled with the methylanthraniloyl probe via the 2' or 3' sugar hydroxyls, has an absorption which overlaps with the UV-Vis features of many metal-binding proteins. However, by exploiting analogs like BODIPY-FL and trinitrophenyl-labeled nucleotides which have better photophysical properties for metalloprotein applications, these approaches have the potential to reveal the mechanistic underpinnings of NTPases required for metallocluster biosynthesis.

Identifying the Protein Interactions of the Cytosolic Iron-Sulfur Cluster Targeting Complex Essential for Its Assembly and Recognition of Apo-Targets.

The cytosolic iron-sulfur cluster assembly (CIA) system assembles iron-sulfur (FeS) cluster cofactors and inserts them into >20 apoprotein targets residing in the cytosol and nucleus. Three CIA proteins, called Cia1, Cia2, and Met18 in yeast, form the targeting complex responsible for apo-target recognition. There is little information about the structure of this complex or its mechanism of CIA substrate recognition. Herein, we exploit affinity co-purification and size exclusion chromatography to determine the subunit connectivity and stoichiometry of the CIA targeting complex. We conclude that Cia2 is the organizing center of the targeting complex, which contains one Met18, two Cia1, and four Cia2 polypeptides. To probe target recognition specificity, we utilize the CIA substrates Leu1 and Rad3 as well as the Escherichia coli FeS-binding transcription factor FNR (fumerate nitrate reductase). We demonstrate that both of the yeast CIA substrates are recognized, whereas the bacterial protein is not. Thus, while the targeting complex exhibits flexible target recognition in vitro, it cannot promiscuously recognize any FeS protein. Additionally, we demonstrate that the full CIA targeting complex is required to stably bind Leu1 in vitro, whereas the Met18-Cia2 subcomplex is sufficient to recognize Rad3. Together, these results allow us to propose a unifying model for the architecture of this highly conserved complex and demonstrate what component or subcomplexes are vital for target identification.

Defining the domains of Cia2 required for its essential function in vivo and in vitro.

The cytosolic iron-sulfur cluster assembly (CIA) system biosynthesizes iron-sulfur (FeS) cluster cofactors for cytosolic and nuclear proteins. The yeast Cia2 protein is the central component of the targeting complex which identifies apo-protein targets in the final step of the pathway. Herein, we determine that Cia2 contains five conserved motifs distributed between an intrinsically disordered N-terminal domain and a C-terminal domain of unknown function 59 (DUF59). The disordered domain is dispensible for binding the other subunits of the targeting complex, Met18 and Cia1, and the apo-target Rad3 in vitro. While in vivo assays reveal that the C-terminal domain is sufficient to support viability, several phenotypic assays indicate that deletion of the N-terminal domain negatively impacts CIA function. We additionally establish that Glu208, located within a conserved motif found only in eukaryotic DUF59 proteins, is important for the Cia1-Cia2 interaction in vitro. In vivo, E208A-Cia2 results in a diminished activity of the cytosolic iron sulfur cluster protein, Leu1 but only modest effects on hydroxyurea or methylmethane sulfonate sensitivity. Finally, we demonstrate that neither of the two highly conserved motifs of the DUF59 domain are vital for any of Cia2's interactions in vitro yet mutation of the DPE motif in the DUF59 domain results in a nonfunctional allele in vivo. Our observation that four of the five highly conserved motifs of Cia2 are dispensable for targeting complex formation and apo-target binding suggests that Cia2 is not simply a protein-protein interaction mediator but it likely possesses an additional, currently cryptic, function during the final cluster insertion step of CIA.

The Yeast Nbp35-Cfd1 Cytosolic Iron-Sulfur Cluster Scaffold Is an ATPase.

Nbp35 and Cfd1 are prototypical members of the MRP/Nbp35 class of iron-sulfur (FeS) cluster scaffolds that function to assemble nascent FeS clusters for transfer to FeS-requiring enzymes. Both proteins contain a conserved NTPase domain that genetic studies have demonstrated is essential for their cluster assembly activity inside the cell. It was recently reported that these proteins possess no or very low nucleotide hydrolysis activity in vitro, and thus the role of the NTPase domain in cluster biogenesis has remained uncertain. We have reexamined the NTPase activity of Nbp35, Cfd1, and their complex. Using in vitro assays and site-directed mutagenesis, we demonstrate that the Nbp35 homodimer and the Nbp35-Cfd1 heterodimer are ATPases, whereas the Cfd1 homodimer exhibited no or very low ATPase activity. We ruled out the possibility that the observed ATP hydrolysis activity might result from a contaminating ATPase by showing that mutation of key active site residues reduced activity to background levels. Finally, we demonstrate that the fluorescent ATP analog 2'/3'-O-(N'-methylanthraniloyl)-ATP (mantATP) binds stoichiometrically to Nbp35 with a KD = 15.6 µM and that an Nbp35 mutant deficient in ATP hydrolysis activity also displays an increased KD for mantATP. Together, our results demonstrate that the cytosolic iron-sulfur cluster assembly scaffold is an ATPase and pave the way for interrogating the role of nucleotide hydrolysis in cluster biogenesis by this large family of cluster scaffolding proteins found across all domains of life.

Two distinct mechanisms for TIM barrel prenyltransferases in bacteria.

The reactions of two bacterial TIM barrel prenyltransferases (PTs), MoeO5 and PcrB, were explored. MoeO5, the enzyme responsible for the first step in moenomycin biosynthesis, catalyzes the transfer of farnesyl to 3-phosphoglyceric acid (3PG) to give a product containing a cis-allylic double bond. We show that this reaction involves isomerization to a nerolidyl pyrophosphate intermediate followed by bond rotation prior to attack by the nucleophile. This mechanism is unprecedented for a prenyltransferase that catalyzes an intermolecular coupling. We also show that PcrB transfers geranyl and geranylgeranyl groups to glycerol-1-phosphate (G1P), making it the first known bacterial enzyme to use G1P as a substrate. Unlike MoeO5, PcrB catalyzes prenyl transfer without isomerization to give products that retain the trans-allylic bond of the prenyl donors. The TIM barrel family of PTs is unique in including enzymes that catalyze prenyl transfer by distinctly different reaction mechanisms.

The role of the substrate lipid in processive glycan polymerization by the peptidoglycan glycosyltransferases.

The peptidoglycan glycosyltransferases (PGTs) catalyze the processive polymerization of a C55 lipid-linked disaccharide (Lipid II) to form peptidoglycan, the main component of the bacterial cell wall. Our ability to understand this reaction has been limited due to challenges identifying the appropriate substrate analogues to selectively interrogate the donor (the elongating strand) and acceptor (Lipid II) sites. To address this problem, we have developed an assay using synthetic substrates that can discriminate between the donor and acceptor sites of the PGTs. We have shown that each site has a distinct lipid length preference. We have also established that processive polymerization depends on the length of the lipid attached to the donor.

Studying a cell division amidase using defined peptidoglycan substrates.

Three periplasmic N-acetylmuramoyl-l-alanine amidases are critical for hydrolysis of septal peptidoglycan, which enables cell separation. The amidases cleave the amide bond between the lactyl group of muramic acid and the amino group of l-alanine to release a peptide moiety. Cell division amidases remain largely uncharacterized because substrates suitable for studying them have not been available. Here we have used synthetic peptidoglycan fragments of defined composition to characterize the catalytic activity and substrate specificity of the important Escherichia coli cell division amidase AmiA. We show that AmiA is a zinc metalloprotease that requires at least a tetrasaccharide glycopeptide substrate for cleavage. The approach outlined here can be applied to many other cell wall hydrolases and should enable more detailed studies of accessory proteins proposed to regulate amidase activity in cells.

Complete characterization of the seventeen step moenomycin biosynthetic pathway.

The moenomycins are phosphoglycolipid antibiotics produced by Streptomyces ghanaensis and related organisms. The phosphoglycolipids are the only known active site inhibitors of the peptidoglycan glycosyltransferases, an important family of enzymes involved in the biosynthesis of the bacterial cell wall. Although these natural products have exceptionally potent antibiotic activity, pharmacokinetic limitations have precluded their clinical use. We previously identified the moenomycin biosynthetic gene cluster in order to facilitate biosynthetic approaches to new derivatives. Here, we report a comprehensive set of genetic and enzymatic experiments that establish functions for the 17 moenomycin biosynthetic genes involved in the synthesis of moenomycin and variants. These studies reveal the order of assembly of the full molecular scaffold and define a subset of seven genes involved in the synthesis of bioactive analogues. This work will enable both in vitro and fermentation-based reconstitution of phosphoglycolipid scaffolds so that chemoenzymatic approaches to novel analogues can be explored.

Determination of the in vivo stoichiometry of tyrosyl radical per betabeta' in Saccharomyces cerevisiae ribonucleotide reductase.

The class I ribonucleotide reductases catalyze the conversion of nucleotides to deoxynucleotides and are composed of two subunits: R1 and R2. R1 contains the site for nucleotide reduction and the sites that control substrate specificity and the rate of reduction. R2 houses the essential diferric-tyrosyl radical (Y(*)) cofactor. In Saccharomyces cerevisiae, two R1s, alpha(n) and , have been identified, while R2 is a heterodimer (betabeta'). beta' cannot bind iron and generate the Y(*); consequently, the maximum amount of Y(*) per betabeta' is 1. To determine the cofactor stoichiometry in vivo, a FLAG-tagged beta ((FLAG)beta) was constructed and integrated into the genome of Y300 (MHY343). This strain facilitated the rapid isolation of endogenous levels of (FLAG)betabeta' by immunoaffinity chromatography, which was found to have 0.45 +/- 0.08 Y(*)/(FLAG)betabeta' and a specific activity of 2.3 +/- 0.5 micromol min(-1) mg(-1). (FLAG)betabeta' isolated from MMS-treated MHY343 cells or cells containing a deletion of the transcriptional repressor gene CRT1 also gave a Y(*)/(FLAG)betabeta' ratio of 0.5. To determine the Y(*)/betabeta' ratio without R2 isolation, whole cell EPR and quantitative Western blots of beta were performed using different strains and growth conditions. The wild-type (wt) strains gave a Y(*)/betabeta' ratio of 0.83-0.89. The same strains either treated with MMS or containing a crt1Delta gave ratios between 0.49 and 0.72. Nucleotide reduction assays and quantitative Western blots from the same strains provided an independent measure and confirmation of the Y(*)/betabeta' ratios. Thus, under normal growth conditions, the cell assembles stoichiometric amounts of Y(*) and modulation of Y(*) concentration is not involved in the regulation of RNR activity.

Nuclear localization of the Saccharomyces cerevisiae ribonucleotide reductase small subunit requires a karyopherin and a WD40 repeat protein.

Ribonucleotide reductase (RNR) catalyzes the reduction of ribonucleotides to the corresponding deoxyribonucleotides and is an essential enzyme for DNA replication and repair. Cells have evolved intricate mechanisms to regulate RNR activity to ensure high fidelity of DNA replication during normal cell-cycle progression and of DNA repair upon genotoxic stress. The RNR holoenzyme is composed of a large subunit R1 (alpha, oligomeric state unknown) and a small subunit R2 (beta(2)). R1 binds substrates and allosteric effectors; R2 contains a diferric-tyrosyl radical [(Fe)(2)-Y.] cofactor that is required for catalysis. In Saccharomyces cerevisiae, R1 is predominantly localized in the cytoplasm, whereas R2, which is a heterodimer (betabeta'), is predominantly in the nucleus. When cells encounter DNA damage or stress during replication, betabeta' is redistributed from the nucleus to the cytoplasm in a checkpoint-dependent manner, resulting in the colocalization of R1 and R2. We have identified two proteins that have an important role in betabeta' nuclear localization: the importin beta homolog Kap122 and the WD40 repeat protein Wtm1. Deletion of either WTM1 or KAP122 leads to loss of betabeta' nuclear localization. Wtm1 and its paralog Wtm2 are both nuclear proteins that are in the same protein complex with betabeta'. Wtm1 also interacts with Kap122 in vivo and requires Kap122 for its nuclear localization. Our results suggest that Wtm1 acts either as an adaptor to facilitate nuclear import of betabeta' by Kap122 or as an anchor to retain betabeta' in the nucleus.

The active form of the Saccharomyces cerevisiae ribonucleotide reductase small subunit is a heterodimer in vitro and in vivo.

The class I ribonucleotide reductases (RNRs) are composed of two homodimeric subunits: R1 and R2. R2 houses a diferric-tyrosyl radical (Y*) cofactor. Saccharomyces cerevisiae has two R2s: Y2 (beta2) and Y4 (beta'2). Y4 is an unusual R2 because three residues required for iron binding have been mutated. While the heterodimer (betabeta') is thought to be the active form, several rnr4delta strains are viable. To resolve this paradox, N-terminally epitope-tagged beta and beta' were expressed in E. coli or integrated into the yeast genome. In vitro exchange studies reveal that when apo-(His6)-beta2 ((His)beta2) is mixed with beta'2, apo-(His)betabeta' forms quantitatively within 2 min. In contrast, holo-betabeta' fails to exchange with apo-(His)beta2 to form holo-(His)betabeta and beta'2. Isolation of genomically encoded tagged beta or beta' from yeast extracts gave a 1:1 complex of beta and beta', suggesting that betabeta' is the active form. The catalytic activity, protein concentrations, and Y* content of the rnr4delta and wild type (wt) strains were compared to clarify the role of beta' in vivo. The Y* content of rnr4delta is 15-fold less than that of wt, consistent with the observed low activity of rnr4delta extracts (<0.01 nmol min(-1) mg(-1)) versus wt (0.06 +/- 0.01 nmol min(-1) mg(-1)). (FLAG)beta2 isolated from the rnr4delta strain has a specific activity of 2 nmol min(-1) mg(-1), similar to that of reconstituted apo-(His)beta2 (10 nmol min(-1) mg(-1)), but significantly less than holo-(His)betabeta' (approximately 2000 nmol min(-1) mg(-1)). These studies together demonstrate that beta' plays a crucial role in cluster assembly in vitro and in vivo and that the active form of the yeast R2 is betabeta'.

Structures of the yeast ribonucleotide reductase Rnr2 and Rnr4 homodimers.

Class I ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides. Eukaryotic RNRs comprise two subunits, the R1 subunit, which contains substrate and allosteric effector binding sites, and the R2 subunit, which houses a catalytically essential diiron-tyrosyl radical cofactor. In Saccharomyces cerevisiae, there are two variants of the R2 subunit, called Rnr2 and Rnr4. Rnr4 is unique in that it lacks three iron-binding residues conserved in all other R2s. Nevertheless, Rnr4 is required to activate Rnr2, and the functional species in vivo is believed to be a heterodimeric complex between the two proteins. The crystal structures of the Rnr2 and Rnr4 homodimers have been determined and are compared to that of the heterodimer. The homodimers are very similar to the heterodimer and to mouse R2 in overall fold, but there are several key differences. In the Rnr2 homodimer, one of the iron-binding helices, helix alphaB, is not well-ordered. In the heterodimer, interactions with a loop region connecting Rnr4 helices alphaA and alpha3 stabilize this Rnr2 helix, which donates iron ligand Asp 145. Sequence differences between Rnr2 and Rnr4 prevent the same interactions from occurring in the Rnr2 homodimer. These findings provide a structural rationale for why the heterodimer is the preferred complex in vivo. The active-site region in the Rnr4 homodimer reveals interactions not apparent in the heterodimer, supporting previous conclusions that this subunit does not bind iron. When taken together, these results support a model in which Rnr4 stabilizes Rnr2 for cofactor assembly and activity.

Pulsed ELDOR spectroscopy measures the distance between the two tyrosyl dadicals in the R2 subunit of the E. coli ribonucleotide reductase.

Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates (NDPs) to deoxynucleoside diphosphates (dNDPs). This RNR is composed of two homodimeric subunits: R1 and R2. R1 binds the NDPs in the active site, and R2 harbors the essential di-iron tyrosyl radical (Y*) cofactor. In this paper, we used PELDOR, a method that detects weak electron-electron dipolar coupling, to make the first direct measurement of the distance between the two Y*'s on each monomer of R2. In the crystal structure of R2, the Y*'s are reduced to tyrosines, and consequently R2 is inactive. In R2, where the Y*'s assume a well-defined geometry with respect to the protein backbone, the PELDOR method allows measurement of a distance of 33.1 +/- 0.2 A that compares favorably to the distance (32.4 A) between the center of mass of the spin density distribution of each Y* on each R2 monomer from the structure. The experiments provide the first direct experimental evidence for two Y*'s in a single R2 in solution.