Alice Douangamath

Unusual Binding Properties of the SH3 Domain of the Yeast Actin-binding Protein Abp1 STRUCTURAL AND FUNCTIONAL ANALYSIS

Abp1p is an actin-binding protein that plays a central role in the organization of Saccharomyces cerevisiae actin cytoskeleton. By a combination of two-hybrid and phage-display approaches, we have identified six new ligands of the Abp1-SH3 domain. None of these SH3-mediated novel interactions was detected in recent all genome high throughput protein interaction projects. Here we show that the SH3-mediated association of Abp1p with the Ser/Thr kinases Prk1p and Ark1p is essential for their localization to actin cortical patches. The Abp1-SH3 domain has a rather unusual binding specificity, because its target peptides contain the tetrapentapeptide +XXXPXXPX+PXXL with positive charges flanking the polyproline core on both sides. Here we present the structure of the Abp1-SH3 domain solved at 1.3-Å resolution. The peptide-binding pockets in the SH3 domain are flanked by two acidic residues that are uncommon at those positions in the SH3 domain family. We have shown by site-directed mutagenesis that one of these negatively charged side chains may be the key determinant for the preference for non-classical ligands.

Structural Evidence for Ammonia Tunneling Across the (Beta Alpha)(8) Barrel of the Imidazole Glycerol Phosphate Synthase Bienzyme Complex.

Since reactive ammonia is not available under physiological conditions, glutamine is used as a source for the incorporation of nitrogen in a number of metabolic pathway intermediates. The heterodimeric ImGP synthase that links histidine and purine biosynthesis belongs to the family of glutamine amidotransferases in which the glutaminase activity is coupled with a subsequent synthase activity specific for each member of the enzyme family. Its X-ray structure from the hyperthermophile Thermotoga maritima shows that the glutaminase subunit is associated with the N-terminal face of the (beta alpha)(8) barrel cyclase subunit. The complex reveals a putative tunnel for the transfer of ammonia over a distance of 25 A. Although ammonia tunneling has been reported for glutamine amidotransferases, the ImGP synthase has evolved a novel mechanism, which extends the known functional properties of the versatile (beta alpha)(8) barrel fold.

Topography for Independent Binding of α-Helical and PPII-Helical Ligands to a Peroxisomal SH3 Domain.

While the function of most small signaling domains is confined to binary ligand interactions, the peroxisomal Pex13p SH3 domain has the unique capacity of binding to two different ligands, Pex5p and Pex14p. We have used this domain as a model to decipher its structurally independent ligand binding sites. By the combined use of X-ray crystallography, NMR spectroscopy, and circular dichroism, we show that the two ligands bind in unrelated conformations to patches located at opposite surfaces of this SH3 domain. Mutations in the Pex13p SH3 domain that abolish interactions within the Pex13p-Pex5p interface specifically impair PTS1-dependent protein import into yeast peroxisomes.

Structural effects of monovalent anions on polymorphic lysozyme crystals.

Understanding direct salt effects on protein crystal polymorphism is addressed by comparing different crystal forms (triclinic, monoclinic, tetragonal and orthorhombic) for hen, turkey, bob white quail and human lysozymes. Four new structures of hen egg-white lysozyme are reported: crystals grown in the presence of NapTS diffracted to 1.85 A, of NaI to 1.6 A, of NaNO(3) to 1.45 A and of KSCN to 1.63 A. These new structures are compared with previously published structures in order to draw a mapping of the surface of different lysozymes interacting with monovalent anions, such as nitrate, chloride, iodide, bromide and thiocyanate. An analysis of the structural sites of these anions in the various lysozyme structures is presented. This study shows common anion sites whatever the crystal form and the chemical nature of anions, while others seem specific to a given geometry and a particular charge environment induced by the crystal packing.

Gentle, fast and effective crystal soaking by acoustic dispensing

A high-throughput method is described for crystal soaking using acoustic droplet ejection, and its effectiveness is demonstrated.

Using high-throughput in situ plate screening to evaluate the effect of dehydration on protein crystals.

Crystal dehydration is a post-crystallization technique that can potentially improve the diffraction of macromolecular crystals. There are currently several ways of undertaking this process; however, dehydration experiments are often limited in their throughput and require prior manipulation of the samples. In the present study, a novel method is proposed that uses in situ plate screening to assess the effect of dehydration by combining the throughput of 96-well crystallization plates with direct X-ray feedback on crystal diffraction quality.

Application of in situ diffraction in high-throughput structure determination platforms.

Macromolecular crystallography (MX) is the most powerful technique available to structural biologists to visualize in atomic detail the macromolecular machinery of the cell. Since the emergence of structural genomics initiatives, significant advances have been made in all key steps of the structure determination process. In particular, third-generation synchrotron sources and the application of highly automated approaches to data acquisition and analysis at these facilities have been the major factors in the rate of increase of macromolecular structures determined annually. A plethora of tools are now available to users of synchrotron beamlines to enable rapid and efficient evaluation of samples, collection of the best data, and in favorable cases structure solution in near real time. Here, we provide a short overview of the emerging use of collecting X-ray diffraction data directly from the crystallization experiment. These in situ experiments are now routinely available to users at a number of synchrotron MX beamlines. A practical guide to the use of the method on the MX suite of beamlines at Diamond Light Source is given.

The XChemExplorer graphical workflow tool for routine or large-scale protein–ligand structure determination

XChemExplorer is a graphical workflow and data-management tool for the parallel determination of protein–ligand complexes. Its implementation, usage and application are described here.

Structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC118

The structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC188 has been determined at 1.72 Å resolution. The structure was solved by molecular replacement, which identified the functional homodimer in the asymmetric unit. Despite only showing 57% sequence identity to its closest homologue, the structure adopted the typical α and β D-ribose 5-phosphate isomerase fold. Comparison to other related structures revealed high homology in the active site, allowing a model of the substrate-bound protein to be proposed. The determination of the structure was expedited by the use of in situ crystallization-plate screening on beamline I04-1 at Diamond Light Source to identify well diffracting protein crystals prior to routine cryocrystallography.

Rapid covalent-probe discovery by electrophile fragment screening

Covalent probes can display unmatched potency, selectivity and duration of action, however, their discovery is challenging. In principle, fragments that can irreversibly bind their target can overcome the low affinity that limits reversible fragment screening. Such electrophilic fragments were considered non-selective and were rarely screened. We hypothesized that mild electrophiles might overcome the selectivity challenge, and constructed a library of 993 mildly electrophilic fragments. We characterized this library by a new high-throughput thiol-reactivity assay and screened them against ten cysteine-containing proteins. Highly reactive and promiscuous fragments were rare and could be easily eliminated. By contrast, we found selective hits for most targets. Combination with high-throughput crystallography allowed rapid progression to potent and selective probes for two enzymes, the deubiquitinase OTUB2, and the pyrophosphatase NUDT7. No inhibitors were previously known for either. This study highlights the potential of electrophile fragment screening as a practical and efficient tool for covalent ligand discovery.