Abstract:Members of the monothiol glutaredoxin family and members of the BolA-like protein family have recently emerged as specific interacting partners involved in iron-sulfur protein maturation and redox regulation pathways. It is known that human mitochondrial BOLA1 and BOLA3 form [2Fe-2S] cluster-bridged dimeric heterocomplexes with the monothiol glutaredoxin GRX5. The structure and cluster coordination of the two [2Fe-2S] heterocomplexes as well as their molecular function are, however, not defined yet. Experiment… Show more
“…4). It was also possible to obtain a structural model of protein complexes that bind a Fe–S cluster, via an experimentally driven docking approach (exploiting the HADDOCK program [115]), by integrating NMR chemical shift perturbation analysis with other experimental data derived from EPR, NMR tailored to paramagnetic systems and mutagenesis [116]. Fig.…”
Section: The Contribution Of Solution Nmr To Understand Molecular Aspmentioning
confidence: 99%
“…In humans, the functional role of the mitochondrial BOLA1 and BOLA3 proteins is still not clearly defined, but it was found that they form a hetero-dimeric complex with GLRX5 in both apo and [2Fe–2S]-cluster bound states [81]. NMR data, combined with other spectroscopic information, allowed us to obtain an experimentally driven docking model of [2Fe–2S] cluster-bridged dimeric BOLA1–GLRX5 and BOLA3–GLRX5 complexes, showing that the BOLA1–GLRX5 complex coordinates a reduced, Rieske-type [2Fe–2S] + cluster, while an oxidized, ferredoxin-like [2Fe–2S] 2+ cluster is present in the BOLA3–GLRX5 complex [116]. It also appeared that the [2Fe–2S] BOLA1–GLRX5 complex is preferentially formed over the [2Fe–2S] BOLA3–GLRX5 complex, as a result of a higher cluster binding affinity.…”
Section: The Contribution Of Solution Nmr To Understand Molecular Aspmentioning
Iron–sulfur proteins were among the first class of metalloproteins that were actively studied using NMR spectroscopy tailored to paramagnetic systems. The hyperfine shifts, their temperature dependencies and the relaxation rates of nuclei of cluster-bound residues are an efficient fingerprint of the nature and the oxidation state of the Fe–S cluster. NMR significantly contributed to the analysis of the magnetic coupling patterns and to the understanding of the electronic structure occurring in [2Fe–2S], [3Fe–4S] and [4Fe–4S] clusters bound to proteins. After the first NMR structure of a paramagnetic protein was obtained for the reduced E. halophila HiPIP I, many NMR structures were determined for several Fe–S proteins in different oxidation states. It was found that differences in chemical shifts, in patterns of unobserved residues, in internal mobility and in thermodynamic stability are suitable data to map subtle changes between the two different oxidation states of the protein. Recently, the interaction networks responsible for maturing human mitochondrial and cytosolic Fe–S proteins have been largely characterized by combining solution NMR standard experiments with those tailored to paramagnetic systems. We show here the contribution of solution NMR in providing a detailed molecular view of “Fe–S interactomics”. This contribution was particularly effective when protein–protein interactions are weak and transient, and thus difficult to be characterized at high resolution with other methodologies.
“…4). It was also possible to obtain a structural model of protein complexes that bind a Fe–S cluster, via an experimentally driven docking approach (exploiting the HADDOCK program [115]), by integrating NMR chemical shift perturbation analysis with other experimental data derived from EPR, NMR tailored to paramagnetic systems and mutagenesis [116]. Fig.…”
Section: The Contribution Of Solution Nmr To Understand Molecular Aspmentioning
confidence: 99%
“…In humans, the functional role of the mitochondrial BOLA1 and BOLA3 proteins is still not clearly defined, but it was found that they form a hetero-dimeric complex with GLRX5 in both apo and [2Fe–2S]-cluster bound states [81]. NMR data, combined with other spectroscopic information, allowed us to obtain an experimentally driven docking model of [2Fe–2S] cluster-bridged dimeric BOLA1–GLRX5 and BOLA3–GLRX5 complexes, showing that the BOLA1–GLRX5 complex coordinates a reduced, Rieske-type [2Fe–2S] + cluster, while an oxidized, ferredoxin-like [2Fe–2S] 2+ cluster is present in the BOLA3–GLRX5 complex [116]. It also appeared that the [2Fe–2S] BOLA1–GLRX5 complex is preferentially formed over the [2Fe–2S] BOLA3–GLRX5 complex, as a result of a higher cluster binding affinity.…”
Section: The Contribution Of Solution Nmr To Understand Molecular Aspmentioning
Iron–sulfur proteins were among the first class of metalloproteins that were actively studied using NMR spectroscopy tailored to paramagnetic systems. The hyperfine shifts, their temperature dependencies and the relaxation rates of nuclei of cluster-bound residues are an efficient fingerprint of the nature and the oxidation state of the Fe–S cluster. NMR significantly contributed to the analysis of the magnetic coupling patterns and to the understanding of the electronic structure occurring in [2Fe–2S], [3Fe–4S] and [4Fe–4S] clusters bound to proteins. After the first NMR structure of a paramagnetic protein was obtained for the reduced E. halophila HiPIP I, many NMR structures were determined for several Fe–S proteins in different oxidation states. It was found that differences in chemical shifts, in patterns of unobserved residues, in internal mobility and in thermodynamic stability are suitable data to map subtle changes between the two different oxidation states of the protein. Recently, the interaction networks responsible for maturing human mitochondrial and cytosolic Fe–S proteins have been largely characterized by combining solution NMR standard experiments with those tailored to paramagnetic systems. We show here the contribution of solution NMR in providing a detailed molecular view of “Fe–S interactomics”. This contribution was particularly effective when protein–protein interactions are weak and transient, and thus difficult to be characterized at high resolution with other methodologies.
“…15,25,28,29,33,34 Furthermore, [2Fe-2S]-bridged Grx homodimers and [2Fe-2S]-bridged Grx-BolA heterocomplexes can transfer Fe-S clusters to specific apo acceptor proteins. 11,15,18,23,27,33,35,36 Of note, the [2Fe-2S] Grx3-Fra2 complex in S. cerevisiae specifically transfers its cluster to the transcription factor Aft2 when these proteins are mixed.…”
The fission yeast Schizosaccharomyces pombe expresses the CCAAT-binding factor Php4 in response to iron deprivation. Php4 forms a transcription complex with Php2, Php3, and Php5 to repress the expression of iron proteins as a means to economize iron usage. Previous in vivo results demonstrate that the function and location of Php4 are regulated in an iron-dependent manner by the cytosolic CGFS type glutaredoxin Grx4. In this study, we aimed to biochemically define these protein-protein and protein-metal interactions. Grx4 was found to bind a [2Fe-2S] cluster with spectroscopic features similar to other CGFS glutaredoxins. Grx4 and Php4 also copurify as a complex with a [2Fe-2S] cluster that is spectroscopically distinct from the cluster on Grx4 alone. In vitro titration experiments suggest that these Fe-S complexes may not be interconvertible in the absence of additional factors. Furthermore, conserved cysteines in Grx4 (Cys172) and Php4 (Cys221 and Cys227) are necessary for Fe-S cluster binding and stable complex formation. Together, these results show that Grx4 controls Php4 function through binding of a bridging [2Fe-2S] cluster.
“…20,21 The most common role for iron-sulfur proteins is transport, biosynthesis and insertion into the final target proteins of the clusters themselves (tagged as substrate). [22][23][24][25][26] This is the result of both the chemical complexity of the iron-containing clusters, thus requiring elaborate biosynthetic and degradation pathways, and the potential toxicity of free iron ions. The second most common roles for iron-sulfur proteins are structural and regulatory.…”
mentioning
confidence: 99%
“…For the latter, the clusters are transiently bound by various proteins along the biosynthetic pathway, also depending upon the final target for cluster insertion. 25,26,29 The electron transfer capabilities of ironsulfur proteins are important but not the only determinant of the higher abundance in the mitochondrion of iron-sulfur proteins with respect to all iron-proteins.…”
Organisms from all kingdoms of life use iron-proteins in a multitude of functional processes. We applied a bioinformatics approach to investigate the human portfolio of iron-proteins. We separated ironproteins based on the chemical nature of their metal-containing cofactors: individual iron ions, heme cofactors and iron-sulfur clusters. We found that about 2% of human genes encode an iron-protein.Of these, 35% are proteins binding individual iron ions, 48% are heme-binding proteins and 17% are iron-sulfur proteins. More than half of the human iron-proteins have a catalytic function. Indeed, we predict that 6.5% of all human enzymes are iron-dependent. This percentage is quite different for the various enzyme classes. Human oxidoreductases feature the largest fraction of iron-dependent family members (about 37%). The distribution of iron proteins in the various cellular compartments is uneven.In particular, the mitochondrion and the endoplasmic reticulum are enriched in iron-proteins with respect to the average content of the cell. Finally, we observed that genes encoding iron-proteins are more frequently associated to pathologies than the all other human genes on average. The present research provides an extensive overview of iron usage by the human proteome, and highlights several specific features of the physiological role of iron ions in human cells.
Significance to metallomicsIron is one of the most ancient and abundant metal ions in living organisms: it participates in fundamental biological processes, such as photosynthesis, and respiration. It is an essential metal ion for humans. Here, we applied a bioinformatics approach to predict the entire set of human proteins that use iron as cofactor. We found that about 2% of human genes encode an iron-protein. In particular, 35% are proteins binding individual iron ions, 48% are heme-binding proteins and 17% are iron-sulfur proteins. Most of these proteins are enzymes: 37% of the human oxidoreductases need an iron ion to perform their catalytic mechanisms. The analysis of the subcellular location highlighted that some organelles are enriched in iron-proteins, in particular about 7% of the proteins localized in the endoplasmic reticulum and in the mitochondrion bind iron. Finally, our data show that mutations in genes encoding iron-binding proteins are more likely to be associated with pathology than all human genes on average.
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