Model of the Interaction between the NF-κB Inhibitory Protein p100 and the E3 Ubiquitin Ligase ß-TrCP based on NMR and Docking Experiments.

NF-κB is a major transcription factor whose activation is triggered through two main activation pathways: the canonical pathway involving disruption of IκB-α/NF-κB complexes and the alternative pathway whose activation relies on the inducible proteolysis of the inhibitory protein p100. One central step controlling p100 processing consists in the interaction of the E3 ubiquitin ligase ß-TrCP with p100, thereby leading to its ubiquitinylation and subsequent either complete degradation or partial proteolysis by the proteasome. However, the interaction mechanism between p100 and ß-TrCP is still poorly defined. In this work, a diphosphorylated 21-mer p100 peptide model containing the phosphodegron motif was used to characterize the interaction with ß-TrCP by NMR. In parallel, docking simulations were performed in order to obtain a model of the 21P-p100/ß-TrCP complex. Saturation transfer difference (STD) experiments were performed in order to highlight the residues of p100 involved in the interaction with the ß-TrCP protein. These results highlighted the importance of pSer865 and pSer869 residues in the interaction with ß-TrCP and particularly the Tyr867 that fits inside the hydrophobe ß-TrCP cavity with the Arg474 guanidinium group. Four other arginines, Arg285, Arg410, Arg431, and Arg521, were found essential in the stabilization of p100 on the ß-TrCP surface. Importantly, the requirement for these five arginine residues of ß-TrCP for the interaction with p100 was further confirmed in vivo, thereby validating the docking model through a biological approach.

Automatic clustering of docking poses in virtual screening process using self-organizing map.

MOTIVATION: Scoring functions provided by the docking software are still a major limiting factor in virtual screening (VS) process to classify compounds. Score analysis of the docking is not able to find out all active compounds. This is due to a bad estimation of the ligand binding energies. Making the assumption that active compounds should have specific contacts with their target to display activity, it would be possible to discriminate active compounds from inactive ones with careful analysis of interatomic contacts between the molecule and the target. However, compounds clustering is very tedious due to the large number of contacts extracted from the different conformations proposed by docking experiments. RESULTS: Structural analysis of docked structures is processed in three steps: (i) a Kohonen self-organizing map (SOM) training phase using drug-protein contact descriptors followed by (ii) an unsupervised cluster analysis and (iii) a Newick file generation for results visualization as a tree. The docking poses are then analysed and classified quickly and automatically by AuPosSOM (Automatic analysis of Poses using SOM). AuPosSOM can be integrated into strategies for VS currently employed. We demonstrate that it is possible to discriminate active compounds from inactive ones using only mean protein contacts' footprints calculation from the multiple conformations given by the docking software. Chemical structure of the compound and key binding residues information are not necessary to find out active molecules. Thus, contact-activity relationship can be employed as a new VS process. AVAILABILITY: AuPosSOM is available at http://www.aupossom.com.

Bacterial Transferase MraY, a Source of Inspiration towards New Antibiotics.

The bacterial resistance to antibiotics constitutes more than ever a severe public health problem. The enzymes involved in bacterial peptidoglycan biosynthesis are pertinent targets for developing new antibiotics, notably the MraY transferase that is not targeted by any marketed drug. Many research groups are currently working on the study or the inhibition of this enzyme. After a concise overview of the role, mechanism and inhibition of MraY, the structure-activity relationships of 5'-triazole-containing aminoribosyluridine inhibitors, we previously synthetized, will be presented. The recently published MraY X-ray structures allowed us to achieve a molecular virtual high-throughput screening of commercial databases and our in-house library resulting in the identification of promising compounds for the further development of new antibiotics.

Phosphorylation-dependent structure of ATF4 peptides derived from a human ATF4 protein, a member of the family of transcription factors.

ATF4 plays a crucial role in the cellular response to stress and the F-box protein beta-TrCP, the receptor component of the SCF E3 ubiquitin ligase responsible for ATF4 degradation by the proteasome, binds to ATF4, and controls its stability. Association between the two proteins depends on ATF4 phosphorylation of serine residues 219 and 224 present in the context of DpSGXXXpS, which is similar but not identical to the DpSGXXpS motif found in most other substrates of beta-TrCP. We used NMR spectroscopy to analyze the structure of the 23P-ATF4 peptide. The 3D structure of the ligand was determined on the basis of NOESY restraints that provide an hairpin loop structure. In contrast, no ordered structure was observed in the NMR experiments for the nonphosphorylated 23-ATF4 in solution. This structural study provides information, which could be used to study the beta-TrCP receptor-ligand interaction in docking procedure. Docking studies showed that the binding epitope of the ligand, is represented by the DpSGIXXpSXE motif. 23P-ATF4 peptide fits the binding pocket of protein beta-TrCP very well, considering that the DpSGIXXpSXE motif adopts an S-turning conformation contrary to the extended DpSGXXpS motif in the other known beta-TrCP ligands.

NMR studies for identifying phosphopeptide ligands of the HIV-1 protein Vpu binding to the F-box protein beta-TrCP.

The human immunodeficiency virus type 1 (HIV-1) Vpu enhances viral particle release and, its interaction with the ubiquitin ligase SCF-beta-TrCP triggers the HIV-1 receptor CD4 degradation by the proteasome. The interaction between beta-TrCP protein and ligands containing the phosphorylated DpSGXXpS motif plays a key role for the development of severe disease states, such as HIV or cancer. This study examines the binding and conformation of phosphopeptides (P1, LIERAEDpSG and P2, EDpSGNEpSE) from HIV protein Vpu to beta-TrCP with the objective of defining the minimum length of peptide needed for effective binding. The screening step can be analyzed by NMR spectroscopy, in particular, saturation transfer NMR methods clearly identify the residues in the peptide that make direct contact with beta-TrCP protein when bound. An analysis of saturation transfer difference (STD) spectra provided clear evidence that the two peptides efficiently bound beta-TrCP receptor protein. To better characterize the ligand-protein interaction, the bound conformation of the phosphorylated peptides was determined using transferred NOESY methods, which gave rise to a well-defined structure. P1 and P2 can fold in a bend arrangement for the DpSG motif, showing the protons identified by STD-NMR as exposed in close proximity at the molecule surface. Ser phosphorylation allows electrostatic interaction and hydrogen bond with the amino acids of the beta-TrCP binding pocket. The upstream LIER hydrophobic region was also essential in binding to a hydrophobic pocket of the beta-TrCP WD domain. These findings are in good agreement with a recently published X-ray structure of a shorter beta-Catenin fragment with the beta-TrCP complex.

HIV-1 encoded virus protein U (Vpu) solution structure of the 41-62 hydrophilic region containing the phosphorylated sites Ser52 and Ser56.

Degradation of the HIV receptor CD4 by the proteasome, mediated by the HIV-1 protein Vpu, is crucial for the release of fully infectious virions. To promote CD4 degradation Vpu has to be phosphorylated on a motif DSGXXS, which is conserved in several signalling proteins known to be degraded by the proteasome upon phosphorylation. Such phosphorylation is required for the interaction of Vpu with the ubiquitin ligase SCF-beta-TrCP that triggers CD4 degradation by the proteasome. In the present work, we used two peptides of 22 amino acids between residues 41 and 62 of Vpu. Vpu41-62 was predicted to form an alpha-helix-flexible-alpha-helix including the phosphorylation motif DS52GNES56 and Vpu_P41-62 was phosphorylated at the two sites Ser52 and Ser56. We analysed the conformational change induced by the phosphorylation of this peptide on the residues Ser52 and Ser56. Homo- and heteronuclear NMR techniques were used to assess the structural influence of phosphorylation. The spectra of the free peptides, Vpu_P41-62 and Vpu41-62, in both H2O (at pH 3.5 and 7.2) and a 1:1 mixture of H2O and trifluoroethanol were completely assigned by a combined application of several two-dimensional proton NMR methods. Analysis of the short- and medium-range NOE connectivities and of the secondary chemical shifts indicated that the peptide segment (42-49) shows a less well-defined helix propensity. The Vpu_P41-62 domain of residues 50-62 forms a loop with the phosphate group pointing away, a short beta-strand and a flexible extended 'tail' of residues 60-62. Residues 50-60 exhibit alpha-proton NMR secondary chemical shift changes from random coil toward more beta-like structure with the combined (temperature, solvent and pH) NMR and molecular calculation experiments. Differences in this molecular region 50-62 suggest that conformational changes of Vpu_P play an important role in Vpu_P-induced degradation of CD4 molecules.

Contact-based ligand-clustering approach for the identification of active compounds in virtual screening.

Evaluation of docking results is one of the most important problems for virtual screening and in silico drug design. Modern approaches for the identification of active compounds in a large data set of docked molecules use energy scoring functions. One of the general and most significant limitations of these methods relates to inaccurate binding energy estimation, which results in false scoring of docked compounds. Automatic analysis of poses using self-organizing maps (AuPosSOM) represents an alternative approach for the evaluation of docking results based on the clustering of compounds by the similarity of their contacts with the receptor. A scoring function was developed for the identification of the active compounds in the AuPosSOM clustered dataset. In addition, the AuPosSOM efficiency for the clustering of compounds and the identification of key contacts considered as important for its activity, were also improved. Benchmark tests for several targets revealed that together with the developed scoring function, AuPosSOM represents a good alternative to the energy-based scoring functions for the evaluation of docking results.

Structural and functional characterization of Nrf2 degradation by the glycogen synthase kinase 3/ß-TrCP axis.

The transcription factor NF-E2-related factor 2 (Nrf2) is a master regulator of a genetic program, termed the phase 2 response, that controls redox homeostasis and participates in multiple aspects of physiology and pathology. Nrf2 protein stability is regulated by two E3 ubiquitin ligase adaptors, Keap1 and ß-TrCP, the latter of which was only recently reported. Here, two-dimensional (2D) gel electrophoresis and site-directed mutagenesis allowed us to identify two serines of Nrf2 that are phosphorylated by glycogen synthase kinase 3ß (GSK-3ß) in the sequence DSGISL. Nuclear magnetic resonance studies defined key residues of this phosphosequence involved in docking to the WD40 propeller of ß-TrCP, through electrostatic and hydrophobic interactions. We also identified three arginine residues of ß-TrCP that participate in Nrf2 docking. Intraperitoneal injection of the GSK-3 inhibitor SB216763 led to increased Nrf2 and heme oxygenase-1 levels in liver and hippocampus. Moreover, mice with hippocampal absence of GSK-3ß exhibited increased levels of Nrf2 and phase 2 gene products, reduced glutathione, and decreased levels of carbonylated proteins and malondialdehyde. This study establishes the structural parameters of the interaction of Nrf2 with the GSK-3/ß-TrCP axis and its functional relevance in the regulation of Nrf2 by the signaling pathways that impinge on GSK-3.

Transfer-NMR and docking studies identify the binding of the peptide derived from activating transcription factor 4 to protein ubiquitin ligase beta-TrCP. Competition STD-NMR with beta-catenin.

ATF4 plays a crucial role in the cellular response to stress. The E3 ubiquitin ligase, SCF beta-TrCP protein responsible for ATF4 degradation by the proteasome, binds to ATF4 through a DpSGXXXpS phosphorylation motif, which is similar but not identical to the DpSGXXpS motif found in most other substrates of beta-TrCP. NMR studies were performed on the free and bound forms of a peptide derived from this ATF4 motif that enabled the elucidation of the conformation of the ligand complexed to the beta-TrCP protein and its binding mode. Saturation transfer difference (STD) NMR allowed the study of competition for binding to beta-TrCP, between the phosphorylation motifs of ATF4 and beta-catenin, to characterize the ATF4 binding epitope. Docking protocols were performed using the crystal structure of the beta-catenin-beta-TrCP complex as a template and NMR results of the ATF4-beta-TrCP complex. In agreement with the STD results, in order to bind to beta-TrCP, the ATF4 DpSGIXXpSXE motif required the association of two negatively charged areas, in addition to the hydrophobic interaction in the beta-TrCP central channel. Docking studies showed that the ATF4 DpSGIXXpSXE motif fits the binding pocket of beta-TrCP through an S-turning conformation. The distance between the two phosphate groups is 17.8 A, which matched the corresponding distance 17.1 A for the other extended DpSGXXpS motif in the beta-TrCP receptor model. This study identifies the residues of the beta-TrCP receptor involved in ligand recognition. Using a new concept of STD competition experiment, we show that ATF4 competes and inhibits binding of beta-catenin to beta-TrCP.

Structure of the complex between phosphorylated substrates and the SCF beta-TrCP ubiquitin ligase receptor: a combined NMR, molecular modeling, and docking approach.

The binding of phosphorylated peptides to the receptor plays a major role in many basic cellular processes in a variety of pathological states. Human beta-TrCP is a key component of a recently characterized E3 ubiquitin ligase complex that regulates protein degradation through the ubiquitin-dependent proteasome pathway. Docking studies were carried out to explore the structural requirements for the beta-TrCP substrates. Docking studies were performed on the bound conformation of the phosphorylated peptides determined by NMR, whereas the beta-TrCP structure was derived by X-ray from Protein Data Bank. After the docking calculation, during which the peptides were conformationally restrained, the complex presented herein was analyzed in terms of ligand-protein interactions and properties of contacting surfaces. The structural requirements for phosphorylated substrates in interaction with beta-TrCP were explored and compared with experimental data from TRNOESY and STD NMR results. The analysis revealed that the bend of the peptide structures, which is indispensable for beta-TrCP recognition, aligns two charged phosphate groups and a central hydrophobic group in a favorable arrangement that leads to the burial of the peptide surface in the binding cleft upon complexation. Through docking simulations, we have identified different specific binding pockets of beta-TrCP according to the ligand in interaction. These data should be valuable in the rational design of a ligand to be used in therapeutic approaches.

Structural studies on 24P-IkappaBalpha peptide derived from a human IkappaB-alpha protein related to the inhibition of the activity of the transcription factor NF-kappaB.

The IkappaB-alpha protein, inhibitor of the transcription factor nuclear factor-kappaB (NF-kappaB), is a cellular substrate of beta-transducin repeat containing protein (beta-TrCP). beta-TrCP is the F-box protein component of an Skp1/Cul1/F-box (SCF)-type ubiquitin ligase complex. beta-TrCP targets the protein IkappaB-alpha for ubiquitination, followed by proteasome degradation. The SCF-beta-TrCP complex specifically recognizes an IkappaB-alpha peptide containing the DpSGXXpS motif in a phosphorylation-dependent manner. A fragment comprising 24 amino acids residues for the phosphorylated peptide at the two sites Ser32 and Ser36 and thus termed 24P-IkappaBalpha (P-IkappaBalpha21-44) was characterized conformationally by NMR spectroscopy and molecular dynamics simulation. In the free states, 24P-IkappaBalpha exhibits mainly a random coil conformation, although the presence of a nascent bend was detected between residues 30 and 36, flanked by two N- and C-terminal disordered regions. The bound conformation of the phosphorylated IkappaB-alpha peptide was obtained using transfer nuclear Overhauser effect spectroscopy (TRNOESY) experiments. To further elucidate the basis of the beta-TrCP interaction, a complex between 24P-IkappaBalpha peptide and beta-TrCP protein was studied using saturation transfer difference (STD) NMR experiments. The conformation of 24P-IkappaBalpha bound to beta-TrCP presents a bend corresponding to the 31DpSGLDpS36 motif and on both sides N- and C-terminal turn regions (Lys22-Asp31 and Met37-Glu43). The bound structure of the phosphorylated peptide suggests that these domains are crucial for the interaction of the peptide with its receptor showing the protons identified by STD NMR as exposed in close proximity to the beta-TrCP surface.

STD and TRNOESY NMR studies on the conformation of the oncogenic protein beta-catenin containing the phosphorylated motif DpSGXXpS bound to the beta-TrCP protein.

beta-TrCP is the F-box protein component of an Skp1/Cul1/F-box (SCF)-type ubiquitin ligase complex. Biochemical studies have suggested that beta-TrCP targets the oncogenic protein beta-catenin for ubiquitination and followed by proteasome degradation. To further elucidate the basis of this interaction, a complex between a 32-residue peptide from beta-catenin containing the phosphorylated motif DpSGXXpS (P-beta-Cat17-48) and beta-TrCP was studied using Saturation Transfer Difference (STD) Nuclear Magnetic Resonance (NMR) experiments. These experiments make it possible to identify the binding epitope of a ligand at atomic resolution. An analysis of STD spectra provided clear evidence that only a few of the 32 residues receive the largest saturation transfer. In particular, the amide protons of the residues in the phosphorylated motif appear to be in close contact to the amino acids of the beta-TrCP binding pocket. The amide and aromatic protons of the His24 and Trp25 residues also receive a significant saturation transfer. These findings are in keeping with a recently published x-ray structure of a shorter beta-catenin fragment with the beta-TrCP1-Skp1 complex and with the earlier findings from mutagenesis and activity assays. To better characterize the ligand-protein interaction, the bound conformation of the phosphorylated beta-catenin peptide was obtained using TRansfer Nuclear Overhauser Effect SpectroscopY (TRNOESY) experiments. Finally, we obtained the bound structure of the phosphorylated peptide showing the protons identified by STD NMR as exposed in close proximity to the molecule surface.

Epitope mapping of the phosphorylation motif of the HIV-1 protein Vpu bound to the selective monoclonal antibody using TRNOESY and STD NMR spectroscopy.

The conformational preferences of a 22-amino acid peptide (LIDRLIERAEDpSGNEpSEGEISA) that mimics the phosphorylated HIV-1-encoded virus protein U (Vpu) antigen have been investigated by NMR spectroscopy. Degradation of HIV receptor CD4 by the proteasome, mediated by the HIV-1 protein Vpu, is crucial for the release of fully infectious virions. Phosphorylation of Vpu at sites Ser52 and Ser56 on the DSGXXS motif is required for the interaction of Vpu with the ubiquitin ligase SCF(beta)(-TrCP) which triggers CD4 degradation by the proteasome. This motif is conserved in several signaling proteins known to be degraded by the proteasome. The interaction of the P-Vpu(41-62) peptide with its monoclonal antibody has been studied by transferred nuclear Overhauser effect NMR spectroscopy (TRNOESY) and saturation transfer difference NMR (STD NMR) spectroscopy. The peptide was found to adopt a bend conformation upon binding to the antibody; the peptide residues (Asp51-pSer56) forming this bend are recognized by the antibody as demonstrated by STD NMR experiments. The three-dimensional structure of P-Vpu(41-62) in the bound conformation was determined by TRNOESY spectra; the peptide adopts a compact structure in the presence of mAb with formation of several bends around Leu45 and Ile46 and around Ile60 and Ser61, with a tight bend created by the DpS(52)GNEpS(56) motif. STD NMR studies provide evidence for the existence of a conformational epitope containing tandem repeats of phosphoserine motifs. The peptide's epitope is predominantly located in the large bend and in the N-terminal segment, implicating bidentale association. These findings are in excellent agreement with a recently published NMR structure required for the interaction of Vpu with the SCF(beta)(-TrCP) protein.

NMR studies of the phosphorylation motif of the HIV-1 protein Vpu bound to the F-box protein beta-TrCP.

A protein-protein association regulated by phosphorylation of serine is examined by NMR studies. Degradation of the HIV receptor CD4 by the proteasome, mediated by the HIV-1 protein Vpu, is crucial for the release of fully infectious virions. Phosphorylation of Vpu at two sites, Ser52 and Ser56, on the motif DSGXXS is required for the interaction of Vpu with the ubiquitin ligase SCF-betaTrCP which triggers CD4 degradation by the proteasome. This motif is conserved in several signaling proteins known to be degraded by the proteasome. To elucidate the basis of beta-TrCP recognition, the bound conformation of the P-Vpu(41-62) peptide was determined by using NMR and MD. The TRNOE intensities provided distance constraints which were used in simulated annealing. The beta-TrCP-bound structure of P-Vpu was found to be similar to the structure of the free peptide in solution and to the structure recognized by its antibody. Residues 50-57 formed a bend while the phosphate groups are pointing away. The binding fragment was studied by STD-NMR spectroscopy. The phosphorylated motif DpS(52)GNEpS(56) was found to make intimate contact with beta-TrCP, and pSer52 displays the strongest binding effect. It is suggested that Ser phosphorylation allows protein-protein association by electrostatic stabilization: an obvious negative binding region of Vpu was recognizable by positive residues (Arg and Lys) of the WD domain of beta-TrCP. The Ile46 residue was also found essential for interaction with the beta-TrCP protein. Leu45 and Ile46 side chains lie in close proximity to a hydrophobic pocket of the WD domain.