Françoise Guerlesquin

Protein–protein interaction inhibition (2P2I) combining high throughput and virtual screening: Application to the HIV-1 Nef protein

Protein–protein recognition is the cornerstone of multiple cellular and pathological functions. Therefore, protein–protein interaction inhibition (2P2I) is endowed with great therapeutic potential despite the initial belief that 2P2I was refractory to small-molecule intervention. Improved knowledge of complex molecular binding surfaces has recently stimulated renewed interest for 2P2I, especially after identification of “hot spots” and first inhibitory compounds. However, the combination of target complexity and lack of starting compound has thwarted experimental results and created intellectual barriers. Here we combined virtual and experimental screening when no previously known inhibitors can be used as starting point in a structure-based research program that targets an SH3 binding surface of the HIV type I Nef protein. High-throughput docking and application of a pharmacophoric filter on one hand and search for analogy on the other hand identified drug-like compounds that were further confirmed to bind Nef in the micromolar range (isothermal titration calorimetry), to target the Nef SH3 binding surface (NMR experiments), and to efficiently compete for Nef–SH3 interactions (cell-based assay, GST pull-down). Initial identification of these compounds by virtual screening was validated by screening of the very same library of compounds in the cell-based assay, demonstrating that a significant enrichment factor was attained by the in silico screening. To our knowledge, our results identify the first set of drug-like compounds that functionally target the HIV-1 Nef SH3 binding surface and provide the basis for a powerful discovery process that should help to speed up 2P2I strategies and open avenues for new class of antiviral molecules.

TRF2 promotes, remodels and protects telomeric Holliday junctions

The ability of the telomeric DNA‐binding protein, TRF2, to stimulate t‐loop formation while preventing t‐loop deletion is believed to be crucial to maintain telomere integrity in mammals. However, little is known on the molecular mechanisms behind these properties of TRF2. In this report, we show that TRF2 greatly increases the rate of Holliday junction (HJ) formation and blocks the cleavage by various types of HJ resolving activities, including the newly identified human GEN1 protein. By using potassium permanganate probing and differential scanning calorimetry, we reveal that the basic domain of TRF2 induces structural changes to the junction. We propose that TRF2 contributes to t‐loop stabilisation by stimulating HJ formation and by preventing resolvase cleavage. These findings provide novel insights into the interplay between telomere protection and homologous recombination and suggest a general model in which TRF2 maintains telomere integrity by controlling the turnover of HJ at t‐loops and at regressed replication forks.

Crystal structure of cytochrome c3 from Desulfovibrio desulfuricans Norway at 1.7 A resolution.

The crystal structure of cytochrome c3 (M(r) 13,000) from Desulfovibrio desulfuricans (118 residues, four heme groups) has been crystallographically refined to 1.7 A resolution using a simulated annealing method, based on the structure-model at 2.5 A resolution, already published. The final R-factor for 10,549 reflections was 0.198 covering the range from 5.5 to 1.7 A resolution. The individual temperature factors were refined for a total of 1059 protein atoms, together with 126 bound solvent molecules. The structure has been analyzed with respect to its detailed conformational properties, secondary structure features, temperature factor behaviour, bound solvent sites and heme geometry and ligation. The characteristic secondary structures of the polypeptide chain of this molecule are one extended alpha-helix, a short beta-strand and 13 reverse turns. The four heme groups are located in different structural environments, all highly exposed to solvent. The particular structural features of the heme environments are compared to the four hemes of the cytochrome c3 from Desulfovibrio vulgaris Miyazaki.

Interaction-induced redox switch in the electron transfer complex rusticyanin-cytochrome c(4).

The blue copper protein rusticyanin isolated from the acidophilic proteobacterium Thiobacillus ferrooxidansdisplays a pH-dependent redox midpoint potential with a pK value of 7 on the oxidized form of the protein. The nature of the alterations of optical and EPR spectra observed above the pK value indicated that the redox-linked deprotonation occurs on the ε-nitrogen of the histidine ligands to the copper ion. Complex formation between rusticyanin and its probable electron transfer partner, cytochrome c 4, induced a decrease of rusticyanins redox midpoint potential by more than 100 mV together with spectral changes similar to those observed above the pK value of the free form. Complex formation thus substantially modifies the pK value of the surface-exposed histidine ligand to the copper ion and thereby tunes the redox midpoint potential of the copper site. Comparisons with reports on other blue copper proteins suggest that the surface-exposed histidine ligand is employed as a redox tuning device by many members of this group of soluble electron carriers.

A novel approach for assesing macromolecular complexes combining soft-docking calculations with NMR data

We present a novel and efficient approach for assessing protein–protein complex formation, which combines ab initio docking calculations performed with the protein docking algorithm BiGGER and chemical shift perturbation data collected with heteronuclear single quantum coherence (HSQC) or TROSY nuclear magnetic resonance (NMR) spectroscopy. This method, termed “restrained soft‐docking,” is validated for several known protein complexes. These data demonstrate that restrained soft‐docking extends the size limitations of NMR spectroscopy and provides an alternative method for investigating macromolecular protein complexes that requires less experimental time, effort, and resources. The potential utility of this novel NMR and simulated docking approach in current structural genomic initiatives is discussed.

Crystal structure of a dimeric octaheme cytochrome c3 (Mr 26000) from Desulfovibrio desulfuricans Norway

BACKGROUND The octaheme cytochrome C3 (M(r) 26,000; cc3) from Desulfovibrio desulfuricans Norway is a dimeric cytochrome made up of two identical subunits, each containing four heme groups. It is involved in the redox transfer chain of sulfate-reducing bacteria, which links the periplasmic oxidation of hydrogen to the cytoplasmic reduction of sulfate. The amino-acid sequence of cc3 shows similarities to that of the tetraheme cytochrome c3 (M(r) 13,000; c3) from the same bacteria. Structural analysis of cc3 forms a basis for understanding the precise roles of the multiheme-containing redox proteins and the reason for the presence of several different multiheme cytochromes in one bacterial strain. RESULTS The crystal structure of cytochrome cc3 has been determined at 2.16 A resolution. The subunits display the c3 structural fold with significant amino-acid substitutions, relative to the tetraheme cytochromes c3, in the regions of the dimer interface. The identical subunits are related by a crystallographic twofold axis, with one heme of each subunit in close contact. The overall structure and the environments of the different heme groups are compared with those of the tetraheme cytochromes c3. CONCLUSIONS A common scheme for interactions between these types of cytochrome and their redox partners involves the interaction of a heme crevice, surrounded by positively charged lysine residues, with acidic residues surrounding the redox partners functional group. Despite the relatively acidic character of cytochrome cc3, the crevice of one heme is surrounded by a high number of positively charged residues, in the same manner as has been reported for cytochromes c3. The environment of this heme is formed by four flexible surface loops which are variable in length and orientation in the different c3-type cytochromes although the overall structural folds are very similar. It has been proposed that this region, adapted in topology and charge, is the interaction site for physiological partners and is also most likely to be the interaction site in the dimeric cytochrome cc3.

Structural Model of the Fe-Hydrogenase/Cytochrome C553 Complex Combining Transverse Relaxation-Optimized Spectroscopy Experiments and Soft Docking Calculations.

Fe-hydrogenase is a 54-kDa iron–sulfur enzyme essential for hydrogen cycling in sulfate-reducing bacteria. The x-ray structure of Desulfovibrio desulfuricans Fe-hydrogenase has recently been solved, but structural information on the recognition of its redox partners is essential to understand the structure-function relationships of the enzyme. In the present work, we have obtained a structural model of the complex of Fe-hydrogenase with its redox partner, the cytochrome c 553, combining docking calculations and NMR experiments. The putative models of the complex demonstrate that the small subunit of the hydrogenase has an important role in the complex formation with the redox partner; 50% of the interacting site on the hydrogenase involves the small subunit. The closest contact between the redox centers is observed between Cys-38, a ligand of the distal cluster of the hydrogenase and Cys-10, a ligand of the heme in the cytochrome. The electron pathway from the distal cluster of the Fe-hydrogenase to the heme of cytochromec 553 was investigated using the software Greenpath and indicates that the observed cysteine/cysteine contact has an essential role. The spatial arrangement of the residues on the interface of the complex is very similar to that already described in the ferredoxin-cytochrome c 553 complex, which therefore, is a very good model for the interacting domain of the Fe-hydrogenase-cytochrome c 553.

Comparative studies of two ferredoxins from Desulfovibrio desulfuricans Norway

Two ferredoxins isolated from Desulfovibrio desulfuricans Norway have been purified and characterized. The less acidic, designated as ferredoxin I, contains four iron atoms, four acid-labile sulfur groups and six cysteine residues per molecule. Ferredoxin II is more acidic and abundant than ferredoxin I, but is very unstable to O2. Ferredoxin I and ferredoxin II differ according to amino acid composition but are homologous with respect to their N-terminal amino acid sequence. The absorption spectra of the two ferredoxins are similar to those of other Desulfovibrio species. Both proteins appear to be dimers of identical 6000-dalton subunits. Their activity was tested in two types of reaction in the electron transfer chain (phosphoroclastic reaction and sulfite reductase activity). The isolation of two different ferredoxins from the same organism, Desulfovibrio, has been reported in Desulfovibrio africanus but the significance of two ferredoxins functioning in the same electron transfer chain is not yet understood.

Computational Studies of Human Galectin-1: Role of Conserved Tryptophan Residue in Stacking Interaction with Carbohydrate Ligands

Abstract Galectins belong to the family of glycan-binding proteins, defined by at least one conserved carbohydrate-recognition domain with a highly conserved amino acid sequence and affinity for β galactosides. They all possess a tryptophan residue in the carbohydrate binding site that forms hydrophobic contacts with the carbohydrate ligands. Site directed mutagenesis experiments have shown that this conserved aromatic residue plays a key role in the interaction. We have studied the interaction between the corresponding human Galectin-1 in silico mutants and different carbohydrate ligands using molecular dynamics in explicit solvent. The results confirm the importance of the conserved tryptophan residue in the affinity of the ligand and gives further insights into the mode of interaction between lactose derivatives and human Galectin-1.

Rapid kinetic studies of the electron-exchange reaction between cytochrome c3 and ferredoxin from Desulfovibrio desulfuricans Norway strain and their individual reactions with dithionite

Abstract The electron-exchange reaction between Desulfovibrio desulfuricans Norway cytochrome c3 and ferredoxin was investigated by rapid-mixing techniques under anaerobic conditions. Their reduction by dithionite is biphasic when several redox centres are present in the molecule. The observed first-order rate constant of the rapid and slow phases are both attributed to the reduction by the radical anion SO⨪2. The reduction rate constants for cytochrome c3 (107 M−1 · s−1) are higher than for ferredoxin (105 M−1 · s−1) at 5°C. These results are compared with data also obtained with Desulfovibrio vulgaris cytochrome c3 and discussed in terms of kinetic models implying inter- and/or intramolecular electron exchange. The electron-transfer kinetics between reduced cytochrome c3 and oxidized ferredoxin is monophasic with a rate-limit of 160 s−1 at 5°C. The amplitude detected corresponds to the oxidation of one heme per molecule. A reaction mechanism involving the formation of one intermediate complex, followed by an intramolecular electron exchange leading to the oxidation of the heme of lowest oxidation-reduction potential is proposed. The reverse reaction (i.e., mixing the oxidized cytochrome c3 with reduced ferredoxin) is still monophasic and of an apparent second-order character with a rate constant of 7 · 107 M−1 · s−1 at 5°C. At extremely low ferredoxin concentrations the reaction is limited by the rate of the reverse reaction (k− = 150 s−1). The reduction of four hemes is observed. This extent of reduction reveals that the apparent equilibrium constant for the electron-exchange reaction is greater than 1. Nevertheless, the data support the view that a complex is formed rapidly between the two proteins and is followed by a rapid intramolecular transfer of electrons in either direction. The rate constants for the reduction of cytochromes c3 by ferredoxins, both from different species, are also reported. It is concluded that cytochrome c3 is not equally efficient in both directions of the electron exchange. Depending on the relative oxidation-reduction states and concentrations, one or two hemes and four hemes might be involved in the electron exchange with ferredoxin. The influence of oxidation-reduction potentials, electrostatic interactions and charge distribution on the proteins are taken into account for the interpretation of the electron-exchange mechanism.