Annick Dejaegere

The “Phantom Effect” of the Rexinoid LG100754: Structural and Functional Insights

Retinoic acid receptors (RARs) and Retinoid X nuclear receptors (RXRs) are ligand-dependent transcriptional modulators that execute their biological action through the generation of functional heterodimers. RXR acts as an obligate dimer partner in many signalling pathways, gene regulation by rexinoids depending on the liganded state of the specific heterodimeric partner. To address the question of the effect of rexinoid antagonists on RAR/RXR function, we solved the crystal structure of the heterodimer formed by the ligand binding domain (LBD) of the RARα bound to its natural agonist ligand (all-trans retinoic acid, atRA) and RXRα bound to a rexinoid antagonist (LG100754). We observed that RARα exhibits the canonical agonist conformation and RXRα an antagonist one with the C-terminal H12 flipping out to the solvent. Examination of the protein-LG100754 interactions reveals that its propoxy group sterically prevents the H12 associating with the LBD, without affecting the dimerization or the active conformation of RAR. Although LG100754 has been reported to act as a ‘phantom ligand’ activating RAR in a cellular context, our structural data and biochemical assays demonstrate that LG100754 mediates its effect as a full RXR antagonist. Finally we show that the ‘phantom ligand effect’ of the LG100754 is due to a direct binding of the ligand to RAR that stabilizes coactivator interactions thus accounting for the observed transcriptional activation of RAR/RXR.

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.

Structure-Based Design of Short Peptide Ligands Binding onto the E. coli Processivity Ring

The multimeric DNA sliding clamps confer high processivity to replicative DNA polymerases and are also binding platforms for various enzymes involved in DNA metabolism. These enzymes interact with the clamp through a small peptide that binds into a hydrophobic pocket which is a potential target for the development of new antibacterial compounds. Starting from a generic heptapeptide, we used a structure-based strategy to improve the design of new peptide ligands. Chemical modifications at specific residues result in a dramatic increase of the interaction as measured by SPR and ITC. The affinity of our best hits was improved by 2 orders of magnitude as compared to the natural ligand, reaching 10(-8) M range. The molecular basis of the interactions was analyzed by solving the co-crystal structures of the most relevant peptides bound to the clamp and reveals how chemical modifications establish new contacts and contributes to an increased affinity of the ligand.

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.

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.

Biophysical Approaches Determining Ligand Binding to Biomolecular Targets

Introduction: The who, where, why questions NMT X-ray Surface plasmon resonance Flourescence Electropspray ionization mass spectrometry Synchrotron circular dicroism Thermal denaturation in presence of a fluorescent dye Calorimetry Molecular modelling Examples of the simultaneous use of several techniques to determine ligand binding

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

BackgroundTransposable 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.MethodsUsing 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.ResultsWe 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-valueu2009<u20090.05) 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 (ρu2009=u2009−0.72) which supports anti-regulation.ConclusionsTex19 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.

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

BACKGROUNDnNuclear 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.nnnMETHODSnWe 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.nnnRESULTSnSeveral 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.nnnCONCLUSIONSnOur analysis calls for a re-examination of the currently accepted GRα homodimer structure and experimental investigations of the alternative architectures.nnnGENERAL SIGNIFICANCEnThis work questions the validity of the currently accepted architecture. This has implications for interpreting physiological data and for therapeutic design pertaining to glucocorticoid research.

Chapter 1:Introduction: The Who, Where, Why Questions

The use of biophysical methods for characterizing ligand binding to macromolecules of biological interest is presented in terms of three questions: Who? Where? Why? The “who” question addresses the problem of identification of the ligands binding to a given target, the “where” question addresses the problem of determining the site of binding, and the “why” questions try to establish the physical (especially thermodynamic) basis of binding. This chapter establishes this framework, which will be followed in the rest of the book.