ABSTRACT
Bacterial cells tightly regulate the intracellular concentrations of essential transition metal ions by deploying a panel of metal-regulated transcriptional repressors and activators that bind to operator-promoter regions upstream of regulated genes. Like other zinc uptake regulator (Zur) proteins, Acinetobacter baumannii Zur represses transcription of its regulon when ZnII is replete and binds more weakly to DNA when ZnII is limiting. Previous studies established that Zur proteins are homodimeric and harbor at least two metal sites per protomer or four per dimer. CdII X-ray absorption spectroscopy (XAS) of the Cd2Zn2 AbZur metalloderivative with CdII bound to the allosteric sites reveals a S(N/O)3 first coordination shell. Site-directed mutagenesis suggests that H89 and C100 from the N-terminal DNA binding domain and H107 and E122 from the C-terminal dimerization domain comprise the regulatory metal site. KZn for this allosteric site is 6.0 (±2.2) × 1012 M-1 with a functional "division of labor" among the four metal ligands. N-terminal domain ligands H89 and C100 contribute far more to KZn than H107 and E122, while C100S AbZur uniquely fails to bind to DNA tightly as measured by an in vitro transcription assay. The heterotropic allosteric coupling free energy, ΔGc, is negative, consistent with a higher KZn for the AbZur-DNA complex and defining a bioavailable ZnII set-point of ≈6 × 10-14 M. Small-angle X-ray scattering (SAXS) experiments reveal that only the wild-type Zn homodimer undergoes allosteric switching, while the C100S AbZur fails to switch. These data collectively suggest that switching to a high affinity DNA-binding conformation involves a rotation/translation of one protomer relative to the other in a way that is dependent on the integrity of C100. We place these findings in the context of other Zur proteins and Fur family repressors more broadly.
Subject(s)
Acinetobacter baumannii , Isoquinolines , Sulfonamides , Acinetobacter baumannii/genetics , Acinetobacter baumannii/metabolism , Bacterial Proteins/chemistry , Binding Sites , Cadmium , Protein Subunits , Scattering, Small Angle , Zinc/metabolism , X-Ray Diffraction , Repressor Proteins/metabolism , Metals , DNA/metabolismABSTRACT
Sequestration of the essential nutrient iron from bacterial invaders that colonize the vertebrate host is a central feature of nutritional immunity and the "fight over transition metals" at the host-pathogen interface. The iron quota for many bacterial pathogens is large, as iron enzymes often make up a significant share of the metalloproteome. Iron enzymes play critical roles in respiration, energy metabolism, and other cellular processes by catalyzing a wide range of oxidation-reduction, electron transfer, and oxygen activation reactions. In this Concept article, we discuss recent insights into the diverse ways that bacterial pathogens acquire this essential nutrient, beyond the well-characterized tris-catecholate FeIII complexes, in competition and cooperation with significant host efforts to cripple these processes. We also discuss pathogen strategies to adapt their metabolism to less-than-optimal iron concentrations, and briefly speculate on what might be an integrated adaptive response to the concurrent limitation of both iron and zinc in the infected host.
Subject(s)
Bacteria/metabolism , Ferric Compounds/metabolism , Host-Pathogen InteractionsABSTRACT
Many iron-sulfur proteins involved in cluster trafficking form [2Fe-2S]-cluster-bridged complexes that are often challenging to characterize because of the inherent instability of the cluster at the interface. Herein, we illustrate the use of fast, online buffer exchange coupled to a native mass spectrometry (OBE nMS) method to characterize [2Fe-2S]-cluster-bridged proteins and their transient cluster-transfer intermediates. The use of this mechanistic and protein-characterization tool is demonstrated with holo glutaredoxinâ 5 (GLRX5) homodimer and holo GLRX5:BolA-like proteinâ 3 (BOLA3) heterodimer. Using the OBE nMS method, cluster-transfer reactions between the holo-dimers and apo-ferredoxin (FDX2) are monitored, and intermediate [2Fe-2S] species, such as (FDX2:GLRX5:[2Fe-2S]:GSH) and (FDX2:BOLA3:GLRX5:[2Fe-2S]:GSH) are detected. The OBE nMS method is a robust technique for characterizing iron-sulfur-cluster-bridged protein complexes and transient iron-sulfur-cluster transfer intermediates.
Subject(s)
Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/metabolism , Mass Spectrometry , Glutaredoxins/chemistry , Glutaredoxins/metabolism , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/metabolism , Protein Multimerization , Protein Structure, QuaternaryABSTRACT
A new class of mitochondrial disease has been identified and characterized as Multiple Mitochondrial Dysfunctions Syndrome (MMDS). Four different forms of the disease have each been attributed to point mutations in proteins involved in iron-sulfur (Fe-S) biosynthesis; in particular, MMDS2 has been associated with the protein BOLA3. To date, this protein has been characterized in vitro concerning its ability to form heterodimeric complexes with two putative Fe-S cluster-binding partners: GLRX5 and NFU. However, BOLA3 has yet to be characterized in its own discrete holo form. Herein we describe procedures to isolate and characterize the human holo BOLA3 protein in terms of Fe-S cluster binding and trafficking and demonstrate that human BOLA3 can form a functional homodimer capable of engaging in Fe-S cluster transfer.
Subject(s)
Iron/chemistry , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/metabolism , Protein Multimerization , Sulfur/chemistry , Apoproteins/chemistry , Apoproteins/metabolism , Humans , Protein Structure, Quaternary , Protein TransportABSTRACT
Human glutaredoxin 5 (Grx5) is one of the core components of the Isc (iron-sulfur cluster) assembly and trafficking machinery, and serves as an intermediary cluster carrier, putatively delivering cluster from the Isu scaffold protein to target proteins. The tripeptide glutathione is intimately involved in this role, providing cysteinyl coordination to the iron center of the Grx5-bound [2Fe-2S] cluster. Grx5 has a well-defined glutathione-binding pocket with protein amino acid residues providing many ionic and hydrogen binding contacts to the bound glutathione. In this report, we investigated the importance of these interactions in cluster chirality and exchange reactivity by systematically perturbing the crucial contacts by use of natural and non-natural amino acid substitutions to disrupt the binding contacts from both the protein and glutathione. Native Grx5 could be reconstituted with all of the glutathione analogs used, as well as other thiol ligands, such as DTT or L-cysteine, by in vitro chemical reconstitution, and the holo proteins were found to transfer [2Fe-2S] cluster to apo ferredoxin 1 at comparable rates. However, the circular dichroism spectra of these derivatives displayed prominent differences that reflect perturbations in local cluster chirality. These studies provided a detailed molecular understanding of glutathione-protein interactions in holo Grx5 that define both cluster spectroscopy and exchange chemistry.
Subject(s)
Glutaredoxins/chemistry , Glutathione/chemistry , Static Electricity , Circular Dichroism , Humans , Hydrogen Bonding , Ligands , Stereoisomerism , Sulfhydryl Compounds/chemistryABSTRACT
Monothiol glutaredoxins (Grx) serve as intermediate cluster carriers in iron-sulfur cluster trafficking. The [2Fe-2S]-bound holo forms of Grx proteins display cysteinyl coordination from exogenous glutathione (GSH), in addition to contact from protein-derived Cys. Herein, we report mechanistic studies that investigate the role of exogenous glutathione in defining cluster chirality, ligand exchange, and the cluster transfer chemistry of Saccharomyces cerevisiae Grx3. Systematic perturbations were introduced to the glutathione-binding site by substitution of conserved charged amino acids that form crucial electrostatic contacts with the glutathione molecule. Native Grx3 could also be reconstituted in the absence of glutathione, with either DTT, BME or free L-cysteine as the source of the exogenous Fe-S ligand contact, while retaining full functional reactivity. The delivery of the [2Fe-2S] cluster to Grx3 from cluster donor proteins such as Isa, Nfu, and a [2Fe-2S](GS)4 complex, revealed that electrostatic contacts are of key importance for positioning the exogenous glutathione that in turn influences the chiral environment of the cluster. All Grx3 derivatives were reconstituted by standard chemical reconstitution protocols and found to transfer cluster to apo ferredoxin 1 (Fdx1) at rates comparable to native protein, even when using DTT, BME or free L-cysteine as a thiol source in place of GSH during reconstitution. Kinetic analysis of cluster transfer from holo derivatives to apo Fdx1 has led to a mechanistic model for cluster transfer chemistry of native holo Grx3, and identification of the likely rate-limiting step for the reaction.
Subject(s)
Glutaredoxins/chemistry , Glutathione/chemistry , Oxidoreductases/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/chemistry , Glutaredoxins/metabolism , Glutathione/metabolism , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/metabolism , Kinetics , Ligands , Oxidoreductases/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , StereoisomerismABSTRACT
Post-transcriptional modification of nucleosides in transfer RNAs (tRNAs) is an important process for accurate and efficient translation of the genetic information during protein synthesis in all domains of life. In particular, specific enzymes catalyze the biosynthesis of sulfur-containing nucleosides, such as the derivatives of 2-thiouridine (s2U), 4-thiouridine (s4U), 2-thiocytidine (s2C), and 2-methylthioadenosine (ms2A), within tRNAs. Whereas the mechanism that has prevailed for decades involved persulfide chemistry, more and more tRNA thiolation enzymes have now been shown to contain a [4Fe-4S] cluster. This review summarizes the information over the last ten years concerning the biochemical, spectroscopic and structural characterization of [4Fe-4S]-dependent non-redox tRNA thiolation enzymes.
ABSTRACT
Bi-allelic variants in Iron-Sulfur Cluster Scaffold (NFU1) have previously been associated with multiple mitochondrial dysfunctions syndrome 1 (MMDS1) characterized by early-onset rapidly fatal leukoencephalopathy. We report 19 affected individuals from 10 independent families with ultra-rare bi-allelic NFU1 missense variants associated with a spectrum of early-onset pure to complex hereditary spastic paraplegia (HSP) phenotype with a longer survival (16/19) on one end and neurodevelopmental delay with severe hypotonia (3/19) on the other. Reversible or irreversible neurological decompensation after a febrile illness was common in the cohort, and there were invariable white matter abnormalities on neuroimaging. The study suggests that MMDS1 and HSP could be the two ends of the NFU1-related phenotypic continuum.
Subject(s)
Spastic Paraplegia, Hereditary , Humans , Phenotype , Spastic Paraplegia, Hereditary/genetics , Mutation, Missense , Alleles , Iron/metabolism , Carrier Proteins/geneticsABSTRACT
Mitochondrial BOLA1 is known to form a [2Fe-2S] cluster-bridged heterodimeric complex with mitochondrial monothiol glutaredoxin GLRX5; however, the function of this heterodimeric complex is unclear. Some reports suggest redundant roles for BOLA1 and a related protein, BOLA3, with both involved in the maturation of [4Fe-4S] clusters in a subset of mitochondrial proteins. However, a later report on the structure of BOLA1-GLRX5 heterodimeric complex demonstrated a buried cluster environment and predicted a redox role instead of the cluster trafficking role suggested for the BOLA3-GLRX5 heterodimeric complex. Herein, we describe a detailed kinetic study of relative cluster exchange reactivity involving heterodimeric complex of BOLA1 with GLRX5. By the use of CD spectroscopy, it is demonstrated that [2Fe-2S]-bridged BOLA1-GLRX5 can be readily formed by cluster uptake from donors such as ISCU or [2Fe-2S](GS)4 complex, but not from ISCA1 or ISCA2. Rapid holo-formation following delivery from [2Fe-2S](GS)4 supports possible physiological relevance in the cellular labile iron pool. Holo [2Fe-2S] BOLA1-GLRX5 heterodimeric complex is incapable of donating cluster to apo protein acceptors, providing experimental support for a nontrafficking role. Finally, we report the formation and reactivity of the holo [2Fe-2S]-bridged BOLA1 homodimer (lacking a partner GLRX). While the holo-heterodimer is thermodynamically more stable, by contrast the holo BOLA1 homodimer does demonstrate facile cluster exchange reactivity.
Subject(s)
Glutaredoxins/metabolism , Iron-Sulfur Proteins/metabolism , Mitochondrial Proteins/metabolism , Multiprotein Complexes/metabolism , Circular Dichroism , Glutaredoxins/chemistry , Iron-Sulfur Proteins/chemistry , Kinetics , Mitochondrial Proteins/chemistry , Multiprotein Complexes/chemistry , Protein Binding , Protein Multimerization , Protein Structure, Quaternary , SpectrophotometryABSTRACT
Iron-sulfur (Fe-S) cluster biosynthesis involves the action of a variety of functionally distinct proteins, most of which are evolutionarily conserved. Mutations in these Fe-S scaffold and trafficking proteins can cause diseases such as multiple mitochondrial dysfunctions syndrome (MMDS), sideroblastic anemia, and mitochondrial encephalopathy. Herein, we investigate the effect of Ile67Asn substitution in the BOLA3 protein that results in the MMDS2 phenotype. Although the exact functional role of BOLA3 in Fe-S cluster biosynthesis is not known, the [2Fe-2S]-bridged complex of BOLA3 with GLRX5, another Fe-S protein, has been proposed as a viable intermediary cluster carrier to downstream targets. Our investigations reveal that the Ile67Asn substitution impairs the ability of BOLA3 to bind its physiological partner GLRX5, resulting in a failure to form the [2Fe-2S]-bridged complex. Although no drastic structural change in BOLA3 arises from the substitution, as evidenced by wild-type and mutant BOLA3 1H-15N HSQC and ion mobility native mass spectrometry experiments, this substitution appears to influence cluster reconstitution on downstream proteins leading to the disease phenotype. By contrast, substituted derivatives of the holo homodimeric form of BOLA3 are formed and remain active toward cluster exchange.
Subject(s)
Asparagine/chemistry , Glutaredoxins/metabolism , Isoleucine/chemistry , Mitochondrial Diseases/pathology , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Mutation , Asparagine/genetics , Asparagine/metabolism , Glutaredoxins/chemistry , Glutaredoxins/genetics , Humans , Isoleucine/genetics , Isoleucine/metabolism , Mitochondrial Diseases/genetics , Mitochondrial Diseases/metabolism , Mitochondrial Proteins/chemistry , Mutagenesis, Site-Directed , Protein Conformation , Protein MultimerizationABSTRACT
Telomere length determines the replicative capacity of mammalian cells. Successive telomere reduction to a critically short length can lead to cellular senescence that irreversibly prevents cells from further cell division. A series of Cu complexes has been designed as selective artificial nucleases that degrade G-quadruplex telomeric DNA and exhibit selective DNA binding affinity and cleavage reactivity toward G-quadruplex telomeric DNA over duplex DNA. In contrast to protein-based nucleases that usually lack membrane permeability, significant cellular uptake and nuclear localization of these Cu complexes was observed. Rapid telomere reduction of cancer cells was also observed after only 1 day incubation, while the absence of DNA fragmentation indicates a low level of nonselective DNA cleavage. Robust telomere reduction by the designed Cu complexes is an S-phase-specific event that is associated with increased formation of the G-quadruplex structure during DNA replication.
Subject(s)
Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Copper , G-Quadruplexes/drug effects , Organometallic Compounds/chemical synthesis , Organometallic Compounds/pharmacology , Telomere Shortening/drug effects , Cell Division/drug effects , Cell Line, Tumor , Cellular Senescence/drug effects , Comet Assay , DNA Fragmentation/drug effects , DNA Replication/drug effects , DNA, Neoplasm/drug effects , Drug Design , Humans , Reactive Oxygen Species/metabolism , S Phase/drug effects , Tumor Cells, CulturedABSTRACT
The [2Fe-2S] cluster-bridged complex of BOLA3 with GLRX5 has been implicated in cluster trafficking, but cluster exchange involving this heterocomplex has not been reported. Herein we describe an investigation of the cluster exchange reactivity of holo BOLA3-GLRX complexes using two different monothiol glutaredoxins, H.s. GLRX5 and S.c. Grx3, which share significant identity. We observe that a 1 : 1 mixture of apo BOLA3 and glutaredoxin protein is able to accept a cluster from donors such as ISCU and a [2Fe-2S](GS)4 complex, with preferential formation of the cluster-bridged heterodimer over the plausible holo homodimeric glutaredoxin. Holo BOLA3-GLRX5 transfers clusters to apo acceptors at rates comparable to other Fe-S cluster trafficking proteins. Isothermal titration calorimetry experiments with apo proteins demonstrated a strong binding of BOLA3 with both GLRX5 and Grx3, while binding with an alternative mitochondrial partner, NFU1, was weak. Cluster exchange and calorimetry experiments resulted in a very similar behavior for yeast Grx3 (cytosolic) and human GLRX5 (mitochondrial), indicating conservation across the monothiol glutaredoxin family for interactions with BOLA3 and supporting a functional role for the BOLA3-GLRX5 heterocomplex relative to the previously proposed BOLA3-NFU1 interaction. The results also demonstrate rapid formation of the heterocomplexed holo cluster via delivery from a glutathione-complexed cluster, again indicative of the physiological relevance of the [2Fe-2S](GS)4 complex in the cellular labile iron pool.
Subject(s)
Glutaredoxins/metabolism , Iron-Sulfur Proteins/metabolism , Proteins/metabolism , Carrier Proteins/metabolism , Glutathione/metabolism , Humans , Mitochondrial Proteins , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Non proteinaceous substances are found to be associated with toxic protein aggregates commonly known as fibrils. Hen egg white lysozyme (HEWL) is able to form fibrillar species under various conditions. Here for the first time we report concentration dependent binding affinities of preformed HEWL fibrils towards DNA and RNA at physiological pH (pH 7.4). We have found that HEWL fibrils bind with DNA and RNA that is distinctly different when compared to native HEWL. The association constant (Ka) of native HEWL and ct-DNA at pH 7.4 is 6.8×10(5)M(-1). We have also investigated the conformational alterations of DNA that occur on binding with HEWL fibrils. Our study has demonstrated dominant electrostatic interactions between oppositely charged polyelectrolytes which accounts for the binding of nucleic acids with fibrils. The affinity between the moieties could lead to disruption in the functions of cellular components that might be attributed to the toxicity of the aggregates formed in vivo.