Bacterial Approaches for Assembling Iron-Sulfur Proteins.
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ABSTRACT:
SUBMITTER: Esquilin-Lebron K
PROVIDER: S-EPMC8593673 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:SignificanceIn recent years, bacterial iron-sulfur cluster proteins that function as regulators of gene transcription have emerged as a major new group. In all cases, the cluster acts as a sensor of the environment and enables the organism to adapt to the prevailing conditions. This can range from mounting a response to oxidative or nitrosative stress to switching between anaerobic and aerobic respiratory pathways. The sensitivity of these ancient cofactors to small molecule reactive oxygen and nitrogen species, in particular, makes them ideally suited to function as sensors.Recent advancesAn important challenge is to obtain mechanistic and structural information about how these regulators function and, in particular, how the chemistry occurring at the cluster drives the subsequent regulatory response. For several regulators, including FNR, SoxR, NsrR, IscR, and Wbl proteins, major advances in understanding have been gained recently and these are reviewed here.Critical issuesA common theme emerging from these studies is that the sensitivity and specificity of the cluster of each regulatory protein must be exquisitely controlled by the protein environment of the cluster.Future directionsA major future challenge is to determine, for a range of regulators, the key factors for achieving control of sensitivity/specificity. Such information will lead, eventually, to a system understanding of stress response, which often involves more than one regulator.
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Project description:The miniaturization process applied to rubredoxins generated a class of peptide-based metalloprotein models, named METP (miniaturized electron transfer protein). The crystal structure of Desulfovibrio vulgaris rubredoxin was selected as a template for the construction of a tetrahedral (S(gamma)-Cys)(4) iron-binding site. Analysis of the structure showed that a sphere of 17 A in diameter, centered on the metal, circumscribes two unconnected approximately C(2) symmetry related beta-hairpins, each containing the -Cys-(Aaa)(2)-Cys- sequence. These observations provided a starting point for the design of an undecapeptide, which self assembles in the presence of tetrahedrally coordinating metal ions. The METP peptide was synthesized in good yield by standard methodologies. Successful assembly of the METP peptide with Co(II), Zn(II), Fe(II/III), in the expected 2:1 stoichiometry, was proven by UV-visible and circular dichroism spectroscopies. UV-visible analysis of the metal complexes indicated the four Cys ligands tetrahedrally arrange around the metal ion, as designed. Circular dichroism measurements on both the free and metal-bound forms revealed that the metal coordination drives the peptide chain to fold into a turned conformation. NMR characterization of the Zn(II)-METP complex fully supported the structure of the designed model. These results prove that METP reproduces the main features of rubredoxin.
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Project description:Iron-sulfur clusters act as important cofactors for a number of transcriptional regulators in bacteria, including many mammalian pathogens. The sensitivity of iron-sulfur clusters to iron availability, oxygen tension, and reactive oxygen and nitrogen species enables bacteria to use such regulators to adapt their gene expression profiles rapidly in response to changing environmental conditions. In this review, we discuss how the [4Fe-4S] or [2Fe-2S] cluster-containing regulators FNR, Wbl, aconitase, IscR, NsrR, SoxR, and AirSR contribute to bacterial pathogenesis through control of both metabolism and classical virulence factors. In addition, we briefly review mammalian iron homeostasis as well as oxidative/nitrosative stress to provide context for understanding the function of bacterial iron-sulfur cluster sensors in different niches within the host.
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Project description:Photoinduced charge-transfer dynamics and the influence of cluster size on the dynamics were investigated using five iron-sulfur clusters: the 1Fe-4S cluster in Pyrococcus furiosus rubredoxin, the 2Fe-2S cluster in Pseudomonas putida putidaredoxin, the 4Fe-4S cluster in nitrogenase iron protein, and the 8Fe-7S P-cluster and the 7Fe-9S-1Mo FeMo cofactor in nitrogenase MoFe protein. Laser excitation promotes the iron-sulfur clusters to excited electronic states that relax to lower states. The electronic relaxation lifetimes of the 1Fe-4S, 8Fe-7S, and 7Fe-9S-1Mo clusters are on the picosecond time scale, although the dynamics of the MoFe protein is a mixture of the dynamics of the latter two clusters. The lifetimes of the 2Fe-2S and 4Fe-4S clusters, however, extend to several nanoseconds. A competition between reorganization energies and the density of electronic states (thus electronic coupling between states) mediates the charge-transfer lifetimes, with the 2Fe-2S cluster of Pdx and the 4Fe-4S cluster of Fe protein lying at the optimum leading to them having significantly longer lifetimes. Their long lifetimes make them the optimal candidates for long-range electron transfer and as external photosensitizers for other photoactivated chemical reactions like solar hydrogen production. Potential electron-transfer and hole-transfer pathways that possibly facilitate these charge transfers are proposed.
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Project description:Iron-sulfur clusters may have been the earliest catalytic cofactors on earth, and most modern organisms use them extensively. Although members of the Archaea produce numerous iron-sulfur proteins, the major cluster assembly proteins found in the Bacteria and Eukarya are not universally conserved in archaea. Free-living archaea do have homologs of the bacterial apbC and eukaryotic NBP35 genes that encode iron-sulfur cluster carrier proteins. This study exploits the genetic system of Salmonella enterica to examine the in vivo functionality of apbC/NBP35 homologs from three archaea: Methanococcus maripaludis, Methanocaldococcus jannaschii, and Sulfolobus solfataricus. All three archaeal homologs could correct the tricarballylate growth defect of an S. enterica apbC mutant. Additional genetic studies showed that the conserved Walker box serine and the Cys-X-X-Cys motif of the M. maripaludis MMP0704 protein were both required for function in vivo but that the amino-terminal ferredoxin domain was not. MMP0704 protein and an MMP0704 variant protein missing the N-terminal ferredoxin domain were purified, and the Fe-S clusters were chemically reconstituted. Both proteins bound equimolar concentrations of Fe and S and had UV-visible spectra similar to those of known [4Fe-4S] cluster-containing proteins. This family of dimeric iron-sulfur carrier proteins evolved before the archaeal and eukaryal lineages diverged, representing an ancient mode of cluster assembly.
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Project description:Vaccination has saved billions of human lives and has considerably reduced the economic burden associated with pandemic and endemic infectious diseases. Notwithstanding major advancements in recent decades, multitude diseases remain with no available effective vaccine. While subunit-based vaccines have shown great potential to address the safety concerns of live-attenuated vaccines, their limited immunogenicity remains a major drawback that still needs to be addressed for their use fighting infectious illnesses, autoimmune disorders, and/or cancer. Among the adjuvants and delivery systems for antigens, bacterial proteinaceous supramolecular structures have recently received considerable attention. The use of bacterial proteins with self-assembling properties to deliver antigens offers several advantages, including biocompatibility, stability, molecular specificity, symmetrical organization, and multivalency. Bacterial protein nanoassemblies closely simulate most invading pathogens, acting as an alarm signal for the immune system to mount an effective adaptive immune response. Their nanoscale architecture can be precisely controlled at the atomic level to produce a variety of nanostructures, allowing for infinite possibilities of organized antigen display. For the bottom-up design of the proteinaceous antigen delivery scaffolds, it is essential to understand how the structural and physicochemical properties of the nanoassemblies modulate the strength and polarization of the immune responses. The present review first describes the relationships between structure and the generated immune responses, before discussing potential and current clinical applications.
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Project description:The assembly of long non-contractile phage tails begins with the formation of the tail tip complex (TTC). TTCs are multi-functional protein structures that mediate host cell adsorption and genome injection. The TTC of phage ? is assembled from multiple copies of eight different proteins, including gpL. Purified preparations of gpL and several homologues all displayed a distinct reddish color, suggesting the binding of iron by these proteins. Further characterization of the gpL homologue from phage N15, which was most amenable to in vitro analyses, showed that it contains two domains. The C-terminal domain was demonstrated to coordinate an iron-sulfur cluster, providing the first example of a viral structural protein binding to this type of metal group. We characterized the iron-sulfur cluster using inductively coupled plasma-atomic emission spectroscopy, absorbance spectroscopy, and electron paramagnetic resonance spectroscopy and found that it is an oxygen-sensitive [4Fe-4S](2+) cluster. Four highly conserved cysteine residues were shown to be required for coordinating the iron-sulfur cluster, and substitution of any of these Cys residues with Ser or Ala within the context of ? gpL abolished biological activity. These data imply that the intact iron-sulfur cluster is required for function. The presence of four conserved Cys residues in the C-terminal regions of very diverse gpL homologues suggest that utilization of an iron-sulfur cluster is a widespread feature of non-contractile tailed phages that infect Gram-negative bacteria. In addition, this is the first example of a viral structural protein that binds an iron-sulfur cluster.
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Project description:MitoNEET is a protein of unknown function present in the mitochondrial membrane that was recently shown to bind specifically the antidiabetic drug pioglizatone. Here, we report the crystal structure of the soluble domain (residues 32-108) of human mitoNEET at 1.8-A resolution. The structure reveals an intertwined homodimer, and each subunit was observed to bind a [2Fe-2S] cluster. The [2Fe-2S] ligation pattern of three cysteines and one histidine differs from the known pattern of four cysteines in most cases or two cysteines and two histidines as observed in Rieske proteins. The [2Fe-2S] cluster is packed in a modular structure formed by 17 consecutive residues. The cluster-binding motif is conserved in at least seven distinct groups of proteins from bacteria, archaea, and eukaryotes, which show a consensus sequence of (hb)-C-X(1)-C-X(2)-(S/T)-X(3)-P-(hb)-C-D-X(2)-H, where hb represents a hydrophobic residue; we term this a CCCH-type [2Fe-2S] binding motif. The nine conserved residues in the motif contribute to iron ligation and structure stabilization. UV-visible absorption spectra indicated that mitoNEET can exist in oxidized and reduced states. Our study suggests an electron transfer function for mitoNEET and for other proteins containing the CCCH motif.
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Project description:Assembly of mitochondrial iron-sulfur (Fe/S) proteins is a key process of cells, and defects cause many rare diseases. In the first phase of this pathway, ten Fe/S cluster (ISC) assembly components synthesize and insert [2Fe-2S] clusters. The second phase is dedicated to the assembly of [4Fe-4S] proteins, yet this part is poorly understood. Here, we characterize the BOLA family proteins Bol1 and Bol3 as specific mitochondrial ISC assembly factors that facilitate [4Fe-4S] cluster insertion into a subset of mitochondrial proteins such as lipoate synthase and succinate dehydrogenase. Bol1-Bol3 perform largely overlapping functions, yet cannot replace the ISC protein Nfu1 that also participates in this phase of Fe/S protein biogenesis. Bol1 and Bol3 form dimeric complexes with both monothiol glutaredoxin Grx5 and Nfu1. Complex formation differentially influences the stability of the Grx5-Bol-shared Fe/S clusters. Our findings provide the biochemical basis for explaining the pathological phenotypes of patients with mutations in BOLA3.
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