1-Hydroxy-2(1H)-pyridinone-Based Chelators with Potential Catechol O-Methyl Transferase Inhibition and Neurorescue Dual Action against Parkinson's Disease.

Two analogues of tolcapone where the nitrocatechol group has been replaced by a 1-hydroxy-2(1H)-pyridinone have been designed and synthesised. These compounds are expected to have a dual mode of action both beneficial against Parkinson's disease: they are designed to be inhibitors of catechol O-methyl transferase, which contribute to the reduction of dopamine in the brain, and to protect neurons against oxidative damage. To assess whether these compounds are worthy of biological assessment to demonstrate these effects, measurement of their pKa and stability constants for Fe(III), in silico modelling of their potential to inhibit COMT and blood-brain barrier scoring were performed. These results demonstrate that the compounds may indeed have the desired properties, indicating they are indeed promising candidates for further evaluation.

Agonistic and potentiating effects of perfluoroalkyl substances (PFAS) on the Atlantic cod (Gadus morhua) peroxisome proliferator-activated receptors (Ppars).

Toxicity mediated by per- and polyfluoroalkyl substances (PFAS), and especially perfluoroalkyl acids (PFAAs), has been linked to activation of peroxisome proliferator-activated receptors (Ppar) in many vertebrates. Here, we present the primary structures, phylogeny, and tissue-specific distributions of the Atlantic cod (Gadus morhua) gmPpara1, gmPpara2, gmPparb, and gmPparg, and demonstrate that the carboxylic acids PFHxA, PFOA, PFNA, as well as the sulfonic acid PFHxS, activate gmPpara1 in vitro, which was also supported by in silico analyses. Intriguingly, a binary mixture of PFOA and the non-activating PFOS produced a higher activation of gmPpara1 compared to PFOA alone, suggesting that PFOS has a potentiating effect on receptor activation. Supporting the experimental data, docking and molecular dynamics simulations of single and double-ligand complexes led to the identification of a putative allosteric binding site, which upon binding of PFOS stabilizes an active conformation of gmPpara1. Notably, binary exposures of gmPpara1, gmPpara2, and gmPparb to model-agonists and PFAAs produced similar potentiating effects. This study provides novel mechanistic insights into how PFAAs may modulate the Ppar signaling pathway by either binding the canonical ligand-binding pocket or by interacting with an allosteric binding site. Thus, individual PFAAs, or mixtures, could potentially modulate the Ppar-signaling pathway in Atlantic cod by interfering with at least one gmPpar subtype.

Advances in Vitamin D Receptor Function and Evolution Based on the 3D Structure of the Lamprey Ligand-Binding Domain.

1α,25-dihydroxyvitamin D3 (1,25D3) regulates many physiological processes in vertebrates by binding to the vitamin D receptor (VDR). Phylogenetic analysis indicates that jawless fishes are the most basal vertebrates exhibiting a VDR gene. To elucidate the mechanism driving VDR activation during evolution, we determined the crystal structure of the VDR ligand-binding domain (LBD) complex from the basal vertebratePetromyzon marinus, sea lamprey (lVDR). Comparison of three-dimensional crystal structures of the lVDR-1,25D3 complex with higher vertebrate VDR-1,25D3 structures suggests that 1,25D3 binds to lVDR similarly to human VDR, but with unique features for lVDR around linker regions between H11 and H12 and between H9 and H10. These structural differences may contribute to the marked species differences in transcriptional responses. Furthermore, residue co-evolution analysis of VDR across vertebrates identifies amino acid positions in H9 and the large insertion domain VDR LBD specific as correlated.

Insights into mineralocorticoid receptor homodimerization from a combined molecular modeling and bioinformatics study.

In vertebrates, the mineralocorticoid receptor (MR) is a steroid-activated nuclear receptor (NR) that plays essential roles in water-electrolyte balance and blood pressure homeostasis. It belongs to the group of oxo-steroidian NRs, together with the glucocorticoid (GR), progesterone (PR), and androgen (AR) receptors. Classically, these oxo-steroidian NRs homodimerize and bind to specific genomic sequences to activate gene expression. NRs are multi-domain proteins, and dimerization is mediated by both the DNA (DBD) and ligand binding domains (LBDs), with the latter thought to provide the largest dimerization interface. However, at the structural level, the dimerization of oxo-steroidian receptors LBDs has remained largely a matter of debate and, despite their sequence homology, there is currently no consensus on a common homodimer assembly across the four receptors, that is, GR, PR, AR, and MR. Here, we examined all available MR LBD crystals using different computational methods (protein common interface database, proteins, interfaces, structures and assemblies, protein-protein interaction prediction by structural matching, and evolutionary protein-protein interface classifier, and the molecular mechanics Poisson-Boltzmann surface area method). A consensus is reached by all methods and singles out an interface mediated by helices H9, H10 and the C-terminal F domain as having characteristics of a biologically relevant assembly. Interestingly, a similar assembly was previously identified for GRα, MR closest homolog. Alternative architectures that were proposed for GRα were not observed for MR. These data call for further experimental investigations of oxo-steroid dimer architectures.

Self-Associating Peptides for Modular Bifunctional Conjugation of Tetramer Macromolecules in Living Cells.

Monitoring the assembly of macromolecules to design entities with novel properties can be achieved either chemically creating covalent bonds or by noncovalent connections using appropriate structural motifs. In this report, two self-associating peptides (named K3 and E3) that originate from p53 tetramerization domain were developed as tools for highly specific and noncovalent heterotetramerization of two biomolecules. The pairing/coupling preferences of K3 and E3 were first evaluated by molecular modeling data and confirmed using circular dichroism spectroscopy, size-exclusion chromatography, and biological assays. Regardless of the moieties fused to K3 and E3, these two peptides self-assembled into dimers of dimers to form bivalent heterotetrameric complexes that proved to be extremely stable inside living cells. The benefits of the multivalency in terms of avidity, specificity, and expanded functional activity were strikingly revealed when the proliferating cell nuclear antigen (PCNA), which is essential for DNA replication, was targeted using a heterotetramer presenting both an antibody fragment against PCNA and a specific PCNA binder peptide. In vitro heterotetramerization of these two known PCNA ligands increased their binding efficiencies to PCNA up to 80-fold compared to the best homotetramer counterpart. In cellulo, the heterotetramers were able to efficiently inhibit DNA replication and to trigger cell death. Altogether, we demonstrate that these two biselective self-assembling peptidic domains offer a versatile noncovalent conjugation method that can be easily implemented for protein engineering.

Interaction of a Model Peptide on Gram Negative and Gram Positive Bacterial Sliding Clamps.

Bacterial sliding clamps control the access of DNA polymerases to the replication fork and are appealing targets for antibacterial drug development. It is therefore essential to decipher the polymerase-clamp binding mode across various bacterial species. Here, two residues of the E. coli clamp binding pocket, EcS346 and EcM362, and their cognate residues in M. tuberculosis and B. subtilis clamps, were mutated. The effects of these mutations on the interaction of a model peptide with these variant clamps were evaluated by thermodynamic, molecular dynamics, X-rays crystallography, and biochemical analyses. EcM362 and corresponding residues in Gram positive clamps occupy a strategic position where a mobile residue is essential for an efficient peptide interaction. EcS346 has a more subtle function that modulates the pocket folding dynamics, while the equivalent residue in B. subtilis is essential for polymerase activity and might therefore be a Gram positive-specific molecular marker. Finally, the peptide binds through an induced-fit process to Gram negative and positive pockets, but the complex stability varies according to a pocket-specific network of interactions.

A revisited version of the apo structure of the ligand-binding domain of the human nuclear receptor retinoic X receptor α.

The retinoic X receptor (RXR) plays a crucial role in the superfamily of nuclear receptors (NRs) by acting as an obligatory partner of several nuclear receptors; its role as a transcription factor is thus critical in many signalling pathways, such as metabolism, cell development, differentiation and cellular death. The first published structure of the apo ligand-binding domain (LBD) of RXRα, which is still used as a reference today, contained inaccuracies. In the present work, these inaccuracies were corrected using modern crystallographic tools. The most important correction concerns the presence of a π-bulge in helix H7, which was originally built as a regular α-helix. The presence of several CHAPS molecules, which are visible for the first time in the electron-density map and which stabilize the H1-H3 loop, which contains helix H2, are also revealed. The apo RXR structure has played an essential role in deciphering the molecular mode of action of NR ligands and is still used in numerous biophysical studies. This refined structure should be used preferentially in the future in interpreting experiments as well as for modelling and structural dynamics studies of the apo RXRα LBD.

Allostery in Its Many Disguises: From Theory to Applications.

Allosteric regulation plays an important role in many biological processes, such as signal transduction, transcriptional regulation, and metabolism. Allostery is rooted in the fundamental physical properties of macromolecular systems, but its underlying mechanisms are still poorly understood. A collection of contributions to a recent interdisciplinary CECAM (Center Européen de Calcul Atomique et Moléculaire) workshop is used here to provide an overview of the progress and remaining limitations in the understanding of the mechanistic foundations of allostery gained from computational and experimental analyses of real protein systems and model systems. The main conceptual frameworks instrumental in driving the field are discussed. We illustrate the role of these frameworks in illuminating molecular mechanisms and explaining cellular processes, and describe some of their promising practical applications in engineering molecular sensors and informing drug design efforts.

Recurrent activating mutations of PPARγ associated with luminal bladder tumors.

The upregulation of PPARγ/RXRα transcriptional activity has emerged as a key event in luminal bladder tumors. It renders tumor cell growth PPARγ-dependent and modulates the tumor microenvironment to favor escape from immuno-surveillance. The activation of the pathway has been linked to PPARG gains/amplifications resulting in PPARγ overexpression and to recurrent activating point mutations of RXRα. Here, we report recurrent mutations of PPARγ that also activate the PPARγ/RXRα pathway, conferring PPARγ-dependency and supporting a crucial role of PPARγ in luminal bladder cancer. These mutations are found throughout the protein-including N-terminal, DNA-binding and ligand-binding domains-and most of them enhance protein activity. Structure-function studies of PPARγ variants with mutations in the ligand-binding domain allow identifying structural elements that underpin their gain-of-function. Our study reveals genomic alterations of PPARG that lead to pro-tumorigenic PPARγ/RXRα pathway activation in luminal bladder tumors and may open the way towards alternative options for treatment.

Unrolr: Structural analysis of protein conformations using stochastic proximity embedding.

Molecular dynamics (MD) simulations are widely used to explore the conformational space of biological macromolecules. Advances in hardware, as well as in methods, make the generation of large and complex MD datasets much more common. Although different clustering and dimensionality reduction methods have been applied to MD simulations, there remains a need for improved strategies that handle nonlinear data and/or can be applied to very large datasets. We present an original implementation of the pivot-based version of the stochastic proximity embedding method aimed at large MD datasets using the dihedral distance as a metric. The advantages of the algorithm in terms of data storage and computational efficiency are presented, as well as the implementation realized. Application and testing through the analysis of a 200 ns accelerated MD simulation of a 35-residue villin headpiece is discussed. Analysis of the simulation shows the promise of this method to organize large conformational ensembles. © 2018 Wiley Periodicals, Inc.

Alternative dimerization interfaces in the glucocorticoid receptor-α ligand binding domain.

BACKGROUND: Nuclear hormone receptors (NRs) constitute a large family of multi-domain ligand-activated transcription factors. Dimerization is essential for their regulation, and both DNA binding domain (DBD) and ligand binding domain (LBD) are implicated in dimerization. Intriguingly, the glucocorticoid receptor-α (GRα) presents a DBD dimeric architecture similar to that of the homologous estrogen receptor-α (ERα), but an atypical dimeric architecture for the LBD. The physiological relevance of the proposed GRα LBD dimer is a subject of debate. METHODS: We analyzed all GRα LBD homodimers observed in crystals using an energetic analysis based on the PISA and on the MM/PBSA methods and a sequence conservation analysis, using the ERα LBD dimer as a reference point. RESULTS: Several dimeric assemblies were observed for GRα LBD. The assembly generally taken to be physiologically relevant showed weak binding free energy and no significant residue conservation at the contact interface, while an alternative homodimer mediated by both helix 9 and C-terminal residues showed significant binding free energy and residue conservation. However, none of the GRα LBD assemblies found in crystals are as stable or conserved as the canonical ERα LBD dimer. GRα C-terminal sequence (F-domain) forms a steric obstacle to the canonical dimer assembly in all available structures. CONCLUSIONS: Our analysis calls for a re-examination of the currently accepted GRα homodimer structure and experimental investigations of the alternative architectures. GENERAL SIGNIFICANCE: This work questions the validity of the currently accepted architecture. This has implications for interpreting physiological data and for therapeutic design pertaining to glucocorticoid research.

Structure of the transcription activator target Tra1 within the chromatin modifying complex SAGA.

The transcription co-activator complex SAGA is recruited to gene promoters by sequence-specific transcriptional activators and by chromatin modifications to promote pre-initiation complex formation. The yeast Tra1 subunit is the major target of acidic activators such as Gal4, VP16, or Gcn4 but little is known about its structural organization. The 430 kDa Tra1 subunit and its human homolog the transformation/transcription domain-associated protein TRRAP are members of the phosphatidyl 3-kinase-related kinase (PIKK) family. Here, we present the cryo-EM structure of the entire SAGA complex where the major target of activator binding, the 430 kDa Tra1 protein, is resolved with an average resolution of 5.7 Å. The high content of alpha-helices in Tra1 enabled tracing of the majority of its main chain. Our results highlight the integration of Tra1 within the major epigenetic regulator SAGA.

Allosteric Regulation in the Ligand Binding Domain of Retinoic Acid Receptorγ.

Retinoic acid (RA) plays key roles in cell differentiation and growth arrest through nuclear retinoic acid receptors (RARs), which are ligand-dependent transcription factors. While the main trigger of RAR activation is the binding of RA, phosphorylation of the receptors has also emerged as an important regulatory signal. Phosphorylation of the RARγ N-terminal domain (NTD) is known to play a functional role in neuronal differentiation. In this work, we investigated the phosphorylation of RARγ ligand binding domain (LBD), and present evidence that the phosphorylation status of the LBD affects the phosphorylation of the NTD region. We solved the X-ray structure of a phospho-mimetic mutant of the LBD (RARγ S371E), which we used in molecular dynamics simulations to characterize the consequences of the S371E mutation on the RARγ structural dynamics. Combined with simulations of the wild-type LBD, we show that the conformational equilibria of LBD salt bridges (notably R387-D340) are affected by the S371E mutation, which likely affects the recruitment of the kinase complex that phosphorylates the NTD. The molecular dynamics simulations also showed that a conservative mutation in this salt bridge (R387K) affects the dynamics of the LBD without inducing large conformational changes. Finally, cellular assays showed that the phosphorylation of the NTD of RARγ is differentially regulated by retinoic acid in RARγWT and in the S371N, S371E and R387K mutants. This multidisciplinary work highlights an allosteric coupling between phosphorylations of the LBD and the NTD of RARγ and supports the importance of structural dynamics involving electrostatic interactions in the regulation of RARs activity.

KAT2A/KAT2B-targeted acetylome reveals a role for PLK4 acetylation in preventing centrosome amplification.

Lysine acetylation is a widespread post-translational modification regulating various biological processes. To characterize cellular functions of the human lysine acetyltransferases KAT2A (GCN5) and KAT2B (PCAF), we determined their acetylome by shotgun proteomics. One of the newly identified KAT2A/2B substrate is polo-like kinase 4 (PLK4), a key regulator of centrosome duplication. We demonstrate that KAT2A/2B acetylate the PLK4 kinase domain on residues K45 and K46. Molecular dynamics modelling suggests that K45/K46 acetylation impairs kinase activity by shifting the kinase to an inactive conformation. Accordingly, PLK4 activity is reduced upon in vitro acetylation of its kinase domain. Moreover, the overexpression of the PLK4 K45R/K46R mutant in cells does not lead to centrosome overamplification, as observed with wild-type PLK4. We also find that impairing KAT2A/2B-acetyltransferase activity results in diminished phosphorylation of PLK4 and in excess centrosome numbers in cells. Overall, our study identifies the global human KAT2A/2B acetylome and uncovers that KAT2A/2B acetylation of PLK4 prevents centrosome amplification.

PSSweb: protein structural statistics web server.

With the increasing number of protein structures available, there is a need for tools capable of automating the comparison of ensembles of structures, a common requirement in structural biology and bioinformatics. PSSweb is a web server for protein structural statistics. It takes as input an ensemble of PDB files of protein structures, performs a multiple sequence alignment and computes structural statistics for each position of the alignment. Different optional functionalities are proposed: structure superposition, Cartesian coordinate statistics, dihedral angle calculation and statistics, and a cluster analysis based on dihedral angles. An interactive report is generated, containing a summary of the results, tables, figures and 3D visualization of superposed structures. The server is available at http://pssweb.org.

Quantitative sampling of conformational heterogeneity of a DNA hairpin using molecular dynamics simulations and ultrafast fluorescence spectroscopy.

Molecular dynamics (MD) simulations and time resolved fluorescence (TRF) spectroscopy were combined to quantitatively describe the conformational landscape of the DNA primary binding sequence (PBS) of the HIV-1 genome, a short hairpin targeted by retroviral nucleocapsid proteins implicated in the viral reverse transcription. Three 2-aminopurine (2AP) labeled PBS constructs were studied. For each variant, the complete distribution of fluorescence lifetimes covering 5 orders of magnitude in timescale was measured and the populations of conformers experimentally observed to undergo static quenching were quantified. A binary quantification permitted the comparison of populations from experimental lifetime amplitudes to populations of aromatically stacked 2AP conformers obtained from simulation. Both populations agreed well, supporting the general assumption that quenching of 2AP fluorescence results from pi-stacking interactions with neighboring nucleobases and demonstrating the success of the proposed methodology for the combined analysis of TRF and MD data. Cluster analysis of the latter further identified predominant conformations that were consistent with the fluorescence decay times and amplitudes, providing a structure-based rationalization for the wide range of fluorescence lifetimes. Finally, the simulations provided evidence of local structural perturbations induced by 2AP. The approach presented is a general tool to investigate fine structural heterogeneity in nucleic acid and nucleoprotein assemblies.

Tex19 and Sectm1 concordant molecular phylogenies support co-evolution of both eutherian-specific genes.

BACKGROUND: Transposable elements (TE) have attracted much attention since they shape the genome and contribute to species evolution. Organisms have evolved mechanisms to control TE activity. Testis expressed 19 (Tex19) represses TE expression in mouse testis and placenta. In the human and mouse genomes, Tex19 and Secreted and transmembrane 1 (Sectm1) are neighbors but are not homologs. Sectm1 is involved in immunity and its molecular phylogeny is unknown. METHODS: Using multiple alignments of complete protein sequences (MACS), we inferred Tex19 and Sectm1 molecular phylogenies. Protein conserved regions were identified and folds were predicted. Finally, expression patterns were studied across tissues and species using RNA-seq public data and RT-PCR. RESULTS: We present 2 high quality alignments of 58 Tex19 and 58 Sectm1 protein sequences from 48 organisms. First, both genes are eutherian-specific, i.e., exclusively present in mammals except monotremes (platypus) and marsupials. Second, Tex19 and Sectm1 have both duplicated in Sciurognathi and Bovidae while they have remained as single copy genes in all further placental mammals. Phylogenetic concordance between both genes was significant (p-value < 0.05) and supported co-evolution and functional relationship. At the protein level, Tex19 exhibits 3 conserved regions and 4 invariant cysteines. In particular, a CXXC motif is present in the N-terminal conserved region. Sectm1 exhibits 2 invariant cysteines and an Ig-like domain. Strikingly, Tex19 C-terminal conserved region was lost in Haplorrhini primates while a Sectm1 C-terminal extra domain was acquired. Finally, we have determined that Tex19 and Sectm1 expression levels anti-correlate across the testis of several primates (ρ = -0.72) which supports anti-regulation. CONCLUSIONS: Tex19 and Sectm1 co-evolution and anti-regulated expressions support a strong functional relationship between both genes. Since Tex19 operates a control on TE and Sectm1 plays a role in immunity, Tex19 might suppress an immune response directed against cells that show TE activity in eutherian reproductive tissues.

Molecular dynamics for computational proteomics of methylated histone H3.

BACKGROUND: Post-translational modifications of histones, and in particular of their disordered N-terminal tails, play a major role in epigenetic regulation. The identification of proteins and proteic domains that specifically bind modified histones is therefore of paramount importance to understand the molecular mechanisms of epigenetics. METHODS: We performed an energetic analysis using the MM/PBSA method in order to study known complexes between methylated histone H3 and effector domains of the PHD family. We then developed a simple molecular dynamics based predictive model based on our analysis. RESULTS: We present a thorough validation of our procedure, followed by the computational predictions of new PHD domains specific for binding histone H3 methylated on lysine 4 (K4). CONCLUSIONS: PHD domains recognize methylated K4 on histone H3 in the context of a linear interaction motif (LIM) formed by the first four amino acids of histone H3 as opposed to recognition of a single methylated site. PHD domains with different sequences find chemically equivalent solutions for stabilizing the histone LIM and these can be identified from energetic analysis. This analysis, in turn, allows for the identification of new PHD domains that bind methylated H3K4 using information that cannot be retrieved from sequence comparison alone. GENERAL SIGNIFICANCE: Molecular dynamics simulations can be used to devise computational proteomics protocols that are both easy to implement and interpret, and that yield reliable predictions that compare favorably to and complement experimental proteomics methods. This article is part of a Special Issue entitled Recent developments of molecular dynamics.

Differential modes of peptide binding onto replicative sliding clamps from various bacterial origins.

Bacterial sliding clamps are molecular hubs that interact with many proteins involved in DNA metabolism through their binding, via a conserved peptidic sequence, into a universally conserved pocket. This interacting pocket is acknowledged as a potential molecular target for the development of new antibiotics. We previously designed short peptides with an improved affinity for the Escherichia coli binding pocket. Here we show that these peptides differentially interact with other bacterial clamps, despite the fact that all pockets are structurally similar. Thermodynamic and modeling analyses of the interactions differentiate between two categories of clamps: group I clamps interact efficiently with our designed peptides and assemble the Escherichia coli and related orthologs clamps, whereas group II clamps poorly interact with the same peptides and include Bacillus subtilis and other Gram-positive clamps. These studies also suggest that the peptide binding process could occur via different mechanisms, which depend on the type of clamp.

Functional relationship between matrix metalloproteinase-11 and matrix metalloproteinase-14.

MMP-11 is a key factor in physiopathological tissue remodeling. As an active form is secreted, its activity must be tightly regulated to avoid detrimental effects. Although TIMP-1 and TIMP-2 reversibly inhibit MMP-11, another more drastic scenario, presumably via hydrolysis, could be hypothesized. In this context, we have investigated the possible implication of MMP-14, since it exhibits a spatiotemporal localization similar to MMP-11. Using native HFL1-produced MMP-11 and HT-1080-produced MMP-14 as well as recombinant proteins, we show that MMP-11 is a MMP-14 substrate. MMP-14 cleaves MMP-11 catalytic domain at the PGG(P1)-I(P1')LA and V/IQH(P1)-L(P1')YG scissile bonds, two new cleavage sites. Interestingly, a functional test showed a dramatical reduction in MMP-11 enzymatic activity when incubated with active MMP-14, whereas inactive point-mutated MMP-14 had no effect. This function is conserved between human and mouse. Thus, in addition to the canonical reversible TIMP-dependent inhibitory system, irreversible MMP proteolytic inactivation might occur by cleavage of the catalytic domain in a MMP-dependent manner. Since MMP-14 is produced by HT-1080 cancer cells, whereas MMP-11 is secreted by HFL1 stromal cells, our findings support the emerging importance of tumor-stroma interaction/cross-talk. Moreover, they highlight a Janus-faced MMP-14 function in the MMP cascade, favoring activation of several pro-MMPs, but limiting MMP-11 activity. Finally, both MMPs are active at the cell periphery. Since MMP-14 is present at the cell membrane, whereas MMP-11 is soluble into the cellular microenvironment, this MMP-14 function might represent one critical regulatory mechanism to control the extent of pericellular MMP-11 bioavailability and protect cells from excessive/inappropriate MMP-11 function.