Updated protein domain annotation of the PARP protein family sheds new light on biological function

AlphaFold2 and related computational tools have greatly aided studies of structural biology through their ability to accurately predict protein structures. In the present work, we explored AF2 structural models of the 17 canonical members of the human PARP protein family and supplemented this analysis with new experiments and an overview of recent published data. PARP proteins are typically involved in the modification of proteins and nucleic acids through mono or poly(ADP-ribosyl)ation, but this function can be modulated by the presence of various auxiliary protein domains. Our analysis provides a comprehensive view of the structured domains and long intrinsically disordered regions within human PARPs, offering a revised basis for understanding the function of these proteins. Among other functional insights, the study provides a model of PARP1 domain dynamics in the DNA-free and DNA-bound states and enhances the connection between ADP-ribosylation and RNA biology and between ADP-ribosylation and ubiquitin-like modifications by predicting putative RNA-binding domains and E2-related RWD domains in certain PARPs. In line with the bioinformatic analysis, we demonstrate for the first time PARP14's RNA-binding capability and RNA ADP-ribosylation activity in vitro. While our insights align with existing experimental data and are probably accurate, they need further validation through experiments

Radiation-induced tetramer-to-dimer transition of Escherichia coli lactose repressor

International audienceThe wild type lactose repressor of Escherichia coli is a tetrameric protein formed by two identical dinners. They are associated via a C-terminal 4-helix bundle (called tetramerization domain) whose stability is ensured by the interaction of leucine zipper motifs. Upon in vitro gamma-irradiation the repressor losses its ability to bind the operator DNA sequence due to damage of its DNA-binding domains. Using an engineered dimeric repressor for comparison, we show here that irradiation induces also the change of repressor oligomerisation state from tetramer to dimer. The splitting of the tetramer into dimers can result from the oxidation of the leucine residues of the tetramerization domain

Effects of gamma irradiation on the DNA-protein complex between the estrogen response element and the estrogen receptor

International audienceSignaling by estrogens, risk factors in breast cancer, is mediated through their binding to the estrogen receptor protein (ER), followed by the formation of a complex between ER and a DNA sequence, called estrogen response element (ERE). Anti-estrogens act as competitive inhibitors by blocking the signal transduction. We have studied in vitro the radiosensitivity of the complex between ER alpha, a subtype of this receptor, and a DNA fragment bearing ERE, as well as the influence of an estrogen (estradiol) or an anti-estrogen (tamoxifen) on this radiosensitivity. We observe that the complex is destabilized upon irradiation with gamma rays in aerated aqueous solution. The analysis of the decrease of binding abilities of the two partners shows that destabilization is mainly due to the damage to the protein. The destabilization is reduced when irradiating in presence of tamoxifen and is increased in presence of estradiol. These effects are due to opposite influences of the ligands on the loss of binding ability of ER. The mechanism that can account for our results is: binding of estradiol or tamoxifen induces distinct structural changes of the ER ligand-binding domain that can trigger (by allostery) distinct structural changes of the ER DNA-binding domains and thus, can differently affect ER-ERE interaction

Radiation Damage to a DNA-Binding Protein. Combined Circular Dichroism and Molecular Dynamics Simulation Analysis

International audienceThe E. coli lactose operon, the paradigm of gene expression regulation systems, is the best model for studying the effect of radiation on such systems. The operon function requires the binding of a protein, the repressor, to a specific DNA sequence, the operator. We have previously shown that upon irradiation the repressor loses its operator binding ability. The main radiation-induced lesions of the headpiece have been identified by mass spectrometry. All tyrosine residues are oxidized into 3,4-dihydroxyphenylalanine (DOPA). In the present study we report a detailed characterization of the headpiece radiation-induced modification. An original approach combining circular dichroism measurements and the analysis of molecular dynamics simulation of headpieces bearing DOPA-s instead of tyrosines has been applied. The CD measurements reveal an irreversible modification of the headpiece structure and stability. The molecular dynamics simulation shows a loss of stability shown by an increase in internal dynamics and allows the estimation of the modifications due to tyrosine oxidation for each structural element of the protein. The changes in headpiece structure and stability can explain at least in part the radiation-induced loss of binding ability of the repressor to the operator. This conclusion should hold for all proteins containing radiosensitive amino acids in their DNA-binding site

Oxidation-sensitive residues mediate the DNA-bending activity of MC1 protein

The Methanosarcina thermophila MC1 protein is a small basic protein that is able to bend DNA sharply. When this protein is submitted to oxidative stress through gamma irradiation, it loses its original DNA interaction properties. The protein can still bind DNA but its ability to bend DNA is decreased dramatically. Here, we used different approaches to determine the oxidations that are responsible for this inactivation. Through a combination of proteolysis and mass spectrometry we have identified the three residues that are oxidized preferentially. We show by site directed mutagenesis that two of these residues, Trp74 and Met75, are involved in the DNA binding. Their substitution by alanine leads to a strong reduction in the protein capacity to bend DNA, and a total loss of its ability to recognize bent DNA. Taken together, these results show that oxidation of both these residues is responsible for the protein inactivation. Furthermore, the results confirm the strong relationship between DNA bending and recognition of DNA sequences by the MC1 protein

Modification of DNA radiolysis by DNA-binding proteins: structural aspects

Formation of specific complexes between proteins and their cognate DNA modulates the yields and the location of radiation damage on both partners of the complex. The radiolysis of DNA-protein complexes is studied for: (1) the Escherichia coli lactose operator-repressor complex, (2) the complex between DNA bearing an analogue of an abasic site and the repair protein Fpg of Lactococcus lactis. Experimental patterns of DNA damages are presented and compared to predicted damage distribution obtained using an improved version of the stochastic model RADACK. The same method is used for predicting the location of damages on the proteins. At doses lower than a threshold that depends on the system, proteins protect their specific binding site on DNA while at high doses, the studied complexes are disrupted mainly through protein damage. The loss of binding ability is the functional consequence of the amino-acids modification by OH· radicals. Many of the most probably damaged amino acids are essential for the DNA-protein interaction and within a complex are protected by DNA

Interaction de la protéine NS1 des virus influenza A avec l’ARN : mécanismes impliqués dans la reconnaissance spécifique

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NEMO Trimerizes through Its Coiled-coil C-terminal Domain

While this paper was being revised, the role of the Hsp90 in the assembly of NEMO to the IKK complex was reported (53).International audienceNEMO/IkappaB kinase (IKK) gamma is the regulatory component of the IKK complex comprising the two protein kinases, IKKalpha and IKKbeta. To investigate the self-assembly properties of NEMO and to understand further the mechanism of activation of the IKK complex, we purified wild-type and mutant NEMO expressed in Escherichia coli. In the absence of its IKK partners, recombinant NEMO (rNEMO) is a metastable functional monomer correctly folded, according to its fluorescence and far-UV CD spectra, which is binding specifically to the IKK complex. A minor fraction of rNEMO was found tightly associated with DnaK (E. coli Hsp70). We also examined the interaction of NEMO with prokaryotic and eukaryotic Hsp70, and we showed that the Hsp70-NEMO complex forms a supramolecular structure probably corresponding to an assembly intermediate. In vivo cross-linking experiments indicate that native NEMO in association with IKK is in equilibrium between a dimeric and a trimeric form. Similarly to native NEMO, a NEMO mutant deleted from its IKK binding N-terminal domain (residues 242-388) forms a stable trimeric coiled-coil, suggesting that the association of NEMO with IKK or with Hsp70 prevents incorrect interdomain pairing reactions that could lead to aggregation or to an non-native oligomeric state of rNEMO. We propose a model in which the activation of the IKK complex occurs through the trimerization of NEMO upon binding to a not yet identified upstream activator

Radiationinduced oxidation of proteins in DNA-protein complexes

International audienceA key step in the regulation of gene expression, DNA structuring and DNA repair is the binding of some proteins to specific DNA sequences. Previously we have shown that DNA-binding proteins are acting as efficient protectors against the attack of hydroxyl radicals produced by water radiolysis. They protect their binding sites on DNA by shielding and by radicals scavenging. They also modify the conformation of DNA (compaction, bending) thereby rendering DNA more resistant to radiolysis. But proteins are also vulnerable and get damaged under irradiation. The progressive accumulation of damages on the protein (mainly side chain modifications) firstly affects the configuration of the DNA-protein couple and finally renders the protein unable to play its protective role: the protein loses its ability to bind to its specific DNA sequence. We have studied the effect of irradiation on the E. coli lactose operator-repressor complex. At low doses the protein protects its specific binding site on DNA. At high doses, the complex is disrupted mainly due to the damage to the protein. CD data show that upon irradiation, the structure and the stability of the binding domain of the protein (the headpiece) changes. Fluorescence measurements reveal the degradation of tyrosine residues. Mass spectrometry data complemented by RADACK calculations allow identifying all the oxidized amino-acids of the DNA binding domains of the proteins. Molecular dynamics simulations reveal and characterize the structural changes induced by the oxidation of each amino-acid in the headpiece. Most of the identified oxidized amino-acids are essential for the DNA-protein interaction as revealed by the analysis of the NMR- or crystallography-based structures of the complexes. Thus, irradiation can critically affect proteins properties and consequently can hinder their binding to DNA, due to the oxidation of amino-acids of the DNA-binding domain and to the subsequent conformational changes of it