New insights into the interaction of centrin with Sfi1.

Centrin binds to Rad4(XPC) and Sfi1 through the hydrophobic motif W(1)xxL(4)xxxL(8) in the opposite orientation. Rad4 has one motif, but Sfi1 has approximately 20 repeats, each of which interacts with a centrin molecule. To investigate the parameters involved in centrin binding, we purified a ScSfi1 domain containing 6 repeats complexed with either yeast centrin Cdc31 or human centrin 1. The present study was performed using mutagenesis of centrin and of Sfi1 residues involved in centrin binding and the stability of the centrin-centrin complexes was assessed using thermal denaturation and CD. Calcium stabilized these complexes, as indicated by the Tm increases measured by circular dichroism. The complexes, which were composed of Sfi1 variants and yeast centrin, were analysed in the presence of EDTA. The replacement of W with F within the repeat region yielded a functional repeat (Tm 45°C). The replacement of W with A in two adjacent Sfi1 repeats reduced the thermal stability of the Sfi1-centrin complexes (40°C). We analysed three HsCen1 variants that were homologous to the yeast mutants and induced cell cycle arrest during the G2/M transition. The HsCen1 variants E105K and F113L reduced the thermal stability (50°C, 50°C) of the ScSfi1-HsCen1 complexes; in contrast, the A109T variant exhibited no change in thermal stability relative to the wild-type (60°C). Conversely to ScCdc31, there were no apparent centrin-centrin interactions with wild-type HsCen1, but they did occur for the S170D mutation that mimics PKA phosphorylation at the S170 residue.

Structure-based mutational analysis of ICAT residues mediating negative regulation of ß-catenin co-transcriptional activity.

ICAT (Inhibitor of ß-CAtenin and TCF) is a small acidic protein that negatively regulates ß-catenin co-transcriptional activity by competing with TCF/LEF factors in their binding to ß-catenin superhelical core. In melanoma cells, ICAT competes with LEF1 to negatively regulate the M-MITF and NEDD9 target genes. The structure of ICAT consists of two domains: the 3-helix bundle N-terminal domain binds to ß-catenin Armadillo (Arm) repeats 10-12 and the C-terminal tail binds to Arm repeats 5-9. To elucidate the structural mechanisms governing ICAT/ß-catenin interactions in melanoma cells, three ICAT residues Y15, K19 and V22 in the N-terminal domain, contacting hydrophobic ß-catenin residue F660, were mutated and interaction was assessed by immunoprecipitation. Despite the moderate hydrophobicity of the contact, its removal completely abolished the interaction. In the ICAT C-terminal tail consensus sequence, neutralization of the electrostatic interactions between residues D66, E75 and ß-catenin residues K435, K312, coupled to deletion of the hydrophobic contact between F71 and ß-catenin R386, markedly reduced, but failed to abolish the ICAT-mediated negative regulation of M-MITF and NEDD9 promoters. We conclude that in melanoma cells, anchoring of ICAT N-terminal domain to ß-catenin through the hook made by residue F660, trapped in the pincers formed by ICAT residues Y15 and V22, is crucial for stabilizing the ICAT/ß-catenin complex. This is a prerequisite for binding of the consensus peptide to Arm repeats 5-9 and competition with LEF1. Differences between ICAT and LEF1 in their affinity for ß-catenin may rely on the absence in ICAT of hydrophilic residues between D66 and F71.

Binding of human centrin 2 to the centrosomal protein hSfi1.

hSfi1, a human centrosomal protein with homologs in other eukaryotic organisms, includes 23 repeats, each of 23 amino acids, separated by 10 residue linkers. The main molecular partner in the centrosome is a small, calcium-binding EF-hand protein, the human centrin 2. Using isothermal titration calorimetry experiments, we characterized the centrin-binding capacity of three isolated hSfi1 repeats, two exhibiting the general consensus motif and the third being the unique Pro-containing human repeat. The two standard peptides bind human centrin 2 and its isolated C-terminal domain with high affinity (approximately 10(7) M(-1)) by an enthalpy-driven mechanism, with a moderate Ca2+ dependence. The Pro-containing repeat shows a binding affinity that is two orders of magnitude lower. The target binding site is localized within the C-terminal domain of human centrin 2. Fluorescence titration and NMR spectroscopy show that the well-conserved Trp residue situated in the C-terminus of each repeat is deeply embedded in a protein hydrophobic cavity, indicating that the peptide direction is reversed relative to previously studied centrin targets. The present results suggest that almost all of the repeats of the Sfi1 protein may independently bind centrin molecules. On the basis of this hypothesis and previous studies on centrin self-assembly, we propose a working model for the role of centrin-Sfi1 interactions in the dynamic structure of centrosome-associated contractile fibers.

Benchmark of four popular virtual screening programs: construction of the active/decoy dataset remains a major determinant of measured performance.

BACKGROUND: In a structure-based virtual screening, the choice of the docking program is essential for the success of a hit identification. Benchmarks are meant to help in guiding this choice, especially when undertaken on a large variety of protein targets. Here, the performance of four popular virtual screening programs, Gold, Glide, Surflex and FlexX, is compared using the Directory of Useful Decoys-Enhanced database (DUD-E), which includes 102 targets with an average of 224 ligands per target and 50 decoys per ligand, generated to avoid biases in the benchmarking. Then, a relationship between these program performances and the properties of the targets or the small molecules was investigated. RESULTS: The comparison was based on two metrics, with three different parameters each. The BEDROC scores with α = 80.5, indicated that, on the overall database, Glide succeeded (score > 0.5) for 30 targets, Gold for 27, FlexX for 14 and Surflex for 11. The performance did not depend on the hydrophobicity nor the openness of the protein cavities, neither on the families to which the proteins belong. However, despite the care in the construction of the DUD-E database, the small differences that remain between the actives and the decoys likely explain the successes of Gold, Surflex and FlexX. Moreover, the similarity between the actives of a target and its crystal structure ligand seems to be at the basis of the good performance of Glide. When all targets with significant biases are removed from the benchmarking, a subset of 47 targets remains, for which Glide succeeded for only 5 targets, Gold for 4 and FlexX and Surflex for 2. CONCLUSION: The performance dramatic drop of all four programs when the biases are removed shows that we should beware of virtual screening benchmarks, because good performances may be due to wrong reasons. Therefore, benchmarking would hardly provide guidelines for virtual screening experiments, despite the tendency that is maintained, i.e., Glide and Gold display better performance than FlexX and Surflex. We recommend to always use several programs and combine their results. Graphical AbstractSummary of the results obtained by virtual screening with the four programs, Glide, Gold, Surflex and FlexX, on the 102 targets of the DUD-E database. The percentage of targets with successful results, i.e., with BDEROC(α = 80.5) > 0.5, when the entire database is considered are in Blue, and when targets with biased chemical libraries are removed are in Red.

A chirality change in XPC- and Sfi1-derived peptides affects their affinity for centrin.

The Ca(2+)-binding protein centrin binds to a hydrophobic motif (W(1)xxL(4)xxxL(8)) included in the sequence of several cellular targets: XPC (xeroderma pigmentosum group C protein), Sfi1 (suppressor of fermentation-induced loss of stress resistance protein1), and Sac3 [the central component of the transcription and mRNA export (TREX-2) complex]. However, centrin binding occurs in a reversed orientation (L(8)xxxL(4)xxW(1)) for Sfi1 and Sac3 compared with XPC. Because D-peptides have been investigated for future therapeutic use, we analyzed their centrin-binding properties. Their affinity for centrin was measured using isothermal titration calorimetry. The chirality change in the target-derived peptides affected their ability to bind centrin in a specific manner depending on the sequence orientation of the centrin-binding motif. In contrast to L-XPC-P10, D-XPC-P10 bound C-HsCen1 in a Ca(2+)-dependent manner and to a lesser extent. D-XPC-P10 exhibited a reduced affinity for C-HsCen1 (Ka=0.064 × 10(6) M(-1)) by a factor of 2000 compared with L-XPC-P10 (Ka=132 × 10(6) M(-1)). D-peptides have a lower affinity than L-peptides for centrin, and the strength of this affinity depends on the sequence orientation of the target-derived peptides. The residual affinity observed for D-XPC suggests that the use of d-peptides represents a promising strategy for inhibiting centrin binding to its targets.

vSDC: a method to improve early recognition in virtual screening when limited experimental resources are available.

BACKGROUND: In drug design, one may be confronted to the problem of finding hits for targets for which no small inhibiting molecules are known and only low-throughput experiments are available (like ITC or NMR studies), two common difficulties encountered in a typical academic setting. Using a virtual screening strategy like docking can alleviate some of the problems and save a considerable amount of time by selecting only top-ranking molecules, but only if the method is very efficient, i.e. when a good proportion of actives are found in the 1-10 % best ranked molecules. RESULTS: The use of several programs (in our study, Gold, Surflex, FlexX and Glide were considered) shows a divergence of the results, which presents a difficulty in guiding the experiments. To overcome this divergence and increase the yield of the virtual screening, we created the standard deviation consensus (SDC) and variable SDC (vSDC) methods, consisting of the intersection of molecule sets from several virtual screening programs, based on the standard deviations of their ranking distributions. CONCLUSIONS: SDC allowed us to find hits for two new protein targets by testing only 9 and 11 small molecules from a chemical library of circa 15,000 compounds. Furthermore, vSDC, when applied to the 102 proteins of the DUD-E benchmarking database, succeeded in finding more hits than any of the four isolated programs for 13-60 % of the targets. In addition, when only 10 molecules of each of the 102 chemical libraries were considered, vSDC performed better in the number of hits found, with an improvement of 6-24 % over the 10 best-ranked molecules given by the individual docking programs.Graphical abstractIn drug design, for a given target and a given chemical library, the results obtained with different virtual screening programs are divergent. So how to rationally guide the experimental tests, especially when only a few number of experiments can be made? The variable Standard Deviation Consensus (vSDC) method was developed to answer this issue. Left panel the vSDC principle consists of intersecting molecule sets, chosen on the basis of the standard deviations of their ranking distributions, obtained from various virtual screening programs. In this study Glide, Gold, FlexX and Surflex were used and tested on the 102 targets of the DUD-E database. Right panel Comparison of the average percentage of hits found with vSDC and each of the four programs, when only 10 molecules from each of the 102 chemical libraries of the DUD-E database were considered. On average, vSDC was capable of finding 38 % of the findable hits, against 34 % for Glide, 32 % for Gold, 16 % for FlexX and 14 % for Surflex, showing that with vSDC, it was possible to overcome the unpredictability of the virtual screening results and to improve them.

Structure, dynamics and thermodynamics of the human centrin 2/hSfi1 complex.

Centrin, an EF-hand calcium-binding protein, has been shown to be involved in the duplication of centrosomes, and Sfi1 (Suppressor of fermentation-induced loss of stress resistance protein 1) is one of its centrosomal targets. There are three isoforms of human centrin, but here we only considered centrin 2 (HsCen2). This protein has the ability to bind to any of the approximately 25 repeats of human Sfi1 (hSfi1) with more or less affinity. In this study, we mainly focused on the 17th repeat (R17-hSfi1-20), which presents the highest level of similarity with a well-studied 17-residue peptide (P17-XPC) from human xeroderma pigmentosum complementation group C protein, another centrin target for DNA repair. The only known structure of HsCen2 was resolved in complex with P17-XPC. The 20-residue peptide R17-hSfi1-20 exhibits the motif L8L4W1, which is the reverse of the XPC motif, W1L4L8. Consequently, the dipole of the helix formed by this motif has a reverse orientation. We wished to ascertain the impact of this reversal on the structure, dynamics and affinity of centrin. To address this question, we determined the structure of C-HsCen2 [the C-terminal domain of HsCen2 (T94-Y172)] in complex with R17-hSfi1-20 and monitored its dynamics by NMR, after having verified that the N-terminal domain of HsCen2 does not interact with the peptide. The structure shows that the binding mode is similar to that of P17-XPC. However, we observed a 2 -A translation of the R17-hSfi1-20 helix along its axis, inducing less anchorage in the protein and the disruption of a hydrogen bond between a tryptophan residue in the peptide and a well-conserved nearby glutamate in C-HsCen2. NMR dynamic studies of the complex strongly suggested the existence of an unusual calcium secondary binding mode in calcium-binding loop III, made possible by the uncommon residue composition of this loop. The secondary metal site is only populated at high calcium concentration and depends on the type of bound ligand.

An unusual peptide deformylase features in the human mitochondrial N-terminal methionine excision pathway.

Dedicated machinery for N-terminal methionine excision (NME) was recently identified in plant organelles and shown to be essential in plastids. We report here the existence of mitochondrial NME in mammals, as shown by the identification of cDNAs encoding specific peptide deformylases (PDFs) and new methionine aminopeptidases (MAP1D). We cloned the two full-length human cDNAs and showed that the N-terminal domains of the encoded enzymes were specifically involved in targeting to mitochondria. In contrast to mitochondrial MAP1D, the human PDF sequence differed from that of known PDFs in several key features. We characterized the human PDF fully in vivo and in vitro. Comparison of the processed human enzyme with the plant mitochondrial PDF1A, to which it is phylogenetically related, showed that the human enzyme had an extra N-terminal domain involved in both mitochondrial targeting and enzyme stability. Mammalian PDFs also display non-random substitutions in the conserved motifs important for activity. Human PDF site-directed mutagenesis variants were studied and compared with the corresponding plant PDF1A variants. We found that amino acid substitutions in human PDF specifically altered its catalytic site, resulting in an enzyme intermediate between bacterial PDF1Bs and plant PDF1As. Because (i) human PDF was found to be active both in vitro and in vivo, (ii) the entire machinery is conserved and expressed in most animals, (iii) the mitochondrial genome expresses substrates for these enzymes, and (iv) mRNA synthesis is regulated, we conclude that animal mitochondria have a functional NME machinery that can be regulated.