Olga V. Moroz

JLigand: a graphical tool for the CCP4 template-restraint library

The CCP4 template-restraint library defines restraints for biopolymers, their modifications and ligands that are used in macromolecular structure refinement. JLigand is a graphical editor for generating descriptions of new ligands and covalent linkages.

House cleaning, a part of good housekeeping

Cellular metabolism constantly generates by‐products that are wasteful or even harmful. Such compounds are excreted from the cell or are removed through hydrolysis to normal cellular metabolites by various ‘house‐cleaning’ enzymes. Some of the most important contaminants are non‐canonical nucleoside triphosphates (NTPs) whose incorporation into the nascent DNA leads to increased mutagenesis and DNA damage. Enzymes intercepting abnormal NTPs from incorporation by DNA polymerases work in parallel with DNA repair enzymes that remove lesions produced by modified nucleotides. House‐cleaning NTP pyrophosphatases targeting non‐canonical NTPs belong to at least four structural superfamilies: MutT‐related (Nudix) hydrolases, dUTPase, ITPase (Maf/HAM1) and all‐α NTP pyrophosphatases (MazG). These enzymes have high affinity (Kms in the micromolar range) for their natural substrates (8‐oxo‐dGTP, dUTP, dITP, 2‐oxo‐dATP), which allows them to select these substrates from a mixture containing a ∼1000‐fold excess of canonical NTPs. To date, many house‐cleaning NTPases have been identified only on the basis of their side activity towards canonical NTPs and NDP derivatives. Integration of growing structural and biochemical data on these superfamilies suggests that their new family members cleanse the nucleotide pool of the products of oxidative damage and inappropriate methylation. House‐cleaning enzymes, such as 6‐phosphogluconolactonase, are also part of normal intermediary metabolism. Genomic data suggest that house‐cleaning systems are more abundant than previously thought and include numerous analogous enzymes with overlapping functions. We discuss the structural diversity of these enzymes, their phylogenetic distribution, substrate specificity and the problem of identifying their true substrates.

Both Ca2+ and Zn2+ are essential for S100A12 protein oligomerization and function

BackgroundHuman S100A12 is a member of the S100 family of EF-hand calcium-modulated proteins that are associated with many diseases including cancer, chronic inflammation and neurological disorders. S100A12 is an important factor in host/parasite defenses and in the inflammatory response. Like several other S100 proteins, it binds zinc and copper in addition to calcium. Mechanisms of zinc regulation have been proposed for a number of S100 proteins e.g. S100B, S100A2, S100A7, S100A8/9. The interaction of S100 proteins with their targets is strongly dependent on cellular microenvironment.ResultsThe aim of the study was to explore the factors that influence S100A12 oligomerization and target interaction. A comprehensive series of biochemical and biophysical experiments indicated that changes in the concentration of calcium and zinc led to changes in the oligomeric state of S100A12. Surface plasmon resonance confirmed that the presence of both calcium and zinc is essential for the interaction of S100A12 with one of its extracellular targets, RAGE – the Receptor for Advanced Glycation End products. By using a single-molecule approach we have shown that the presence of zinc in tissue culture medium favors both the oligomerization of exogenous S100A12 protein and its interaction with targets on the cell surface.ConclusionWe have shown that oligomerization and target recognition by S100A12 is regulated by both zinc and calcium. Our present work highlighted the potential role of calcium-binding S100 proteins in zinc metabolism and, in particular, the role of S100A12 in the cross talk between zinc and calcium in cell signaling.

The Crystal Structures of Human S100A12 in Apo Form and in Complex with Zinc: New Insights Into S100A12 Oligomerisation.

The functions of the members of the S100 family of EF-hand proteins are modulated by calcium and, in a number of cases, by zinc or copper. One such protein is S100A12, which is implicated in inflammation and host-parasite responses. Previously, we reported the structures of human S100A12 in both low (dimeric) and high (hexameric) calcium forms and, in addition, that of a complex with copper and calcium. Here we report the crystal structures of the metal-free apo form of human S100A12 at 1.77 A resolution and of the zinc complex in two crystal forms (P2(1)2(1)2(1) and F222) to 1.88 A and 1.73 A resolution, respectively. These are the first structures of a zinc-only complex of an S100 protein to be determined. The zinc complex structure shows significant differences from those of both calcium-loaded and apo-S100A12 structures, and comparisons suggest an explanation for the zinc-induced 1500-fold increase in calcium affinity. In addition, the new structures provide insight into the role of zinc-calcium interplay in the transition of S100A12 from a dimer through a tetramer to a hexamer. The role of both zinc and calcium in target binding by S100A12 during host-parasite responses is confirmed by experiments with paramyosin from the tropical parasites Onchocerca volvulus and Brugia malayi.

The Crystal Structure of the Leishmania major Deoxyuridine Triphosphate Nucleotidohydrolase in Complex with Nucleotide Analogues, dUMP, and Deoxyuridine

Members of the Leishmania genus are the causative agents of the life-threatening disease leishmaniasis. New drugs are being sought due to increasing resistance and adverse side effects with current treatments. The knowledge that dUTPase is an essential enzyme and that the all α-helical dimeric kinetoplastid dUTPases have completely different structures compared with the trimeric β-sheet type dUTPase possessed by most organisms, including humans, make the dimeric enzymes attractive drug targets. Here, we present crystal structures of the Leishmania major dUTPase in complex with substrate analogues, the product dUMP and a substrate fragment, and of the homologous Campylobacter jejuni dUTPase in complex with a triphosphate substrate analogue. The metal-binding properties of both enzymes are shown to be dependent upon the ligand identity, a previously unseen characteristic of this family. Furthermore, structures of the Leishmania enzyme in the presence of dUMP and deoxyuridine coupled with tryptophan fluorescence quenching indicate that occupation of the phosphate binding region is essential for induction of the closed conformation and hence for substrate binding. These findings will aid in the development of dUTPase inhibitors as potential new lead anti-trypanosomal compounds.

The role of zinc in the S100 proteins: insights from the X-ray structures

We here aim to summarise the present knowledge on zinc binding by S100 proteins. While the importance of modulation of the function of the S100 family of EF-hand proteins by calcium is well established, a substantial proportion is also regulated by zinc or copper. Indeed regulation by zinc in addition to calcium was suggested almost as soon as the first S100 protein was discovered and has been confirmed for many family members by numerous experiments. For the first, “His-Zn”, group, zinc-binding sites composed of three histidines and an aspartic acid were first proposed based on sequence comparisons and later confirmed by structural studies. A second, “Cys-Zn”, group lacks such well-defined zinc-binding motifs and for these cysteines were suggested as the main zinc ligands. There is no three-dimensional structure for a Cys-Zn S100 in the presence of zinc. However, analysis of their sequences together with their X-ray structures in the absence of zinc suggests the possibility of two zinc-binding sites: a conserved site with a degree of similarity to those of the His-Zn group and a less-defined site with a Cys interdimer-binding motif. Some S100 protein-mediated events, such as signalling in the extracellular space, where the levels of calcium are already high, are most unlikely to be calcium regulated. Therefore, a broader knowledge of the role of zinc in the functioning of the S100 proteins will add significantly to the understanding how they propagate their signals.

Evidence for Steric Regulation of Fibrinogen Binding to Staphylococcus Aureus Fibronectin-Binding Protein a (Fnbpa).

Background: Staphylococcus aureus fibronectin-binding protein A (FnBPA) binds fibronectin and fibrinogen at adjacent sites. Results: The fibrinogen-binding mechanism is similar but not identical to homologous bacterial proteins. Ternary complex formation by intact fibronectin and fibrinogen on adjacent FnBPA sites could not be demonstrated. Conclusion: Fibrinogen binding is sterically regulated by fibronectin binding. Significance: Steric regulation might result in targeting of S. aureus to fibrin clots. The adjacent fibrinogen (Fg)- and fibronectin (Fn)-binding sites on Fn-binding protein A (FnBPA), a cell surface protein from Staphylococcus aureus, are implicated in the initiation and persistence of infection. FnBPA contains a single Fg-binding site (that also binds elastin) and multiple Fn-binding sites. Here, we solved the structure of the N2N3 domains containing the Fg-binding site of FnBPA in the apo form and in complex with a Fg peptide. The Fg binding mechanism is similar to that of homologous bacterial proteins but without the requirement for “latch” strand residues. We show that the Fg-binding sites and the most N-terminal Fn-binding sites are nonoverlapping but in close proximity. Although Fg and a subdomain of Fn can form a ternary complex on an FnBPA protein construct containing a Fg-binding site and single Fn-binding site, binding of intact Fn appears to inhibit Fg binding, suggesting steric regulation. Given the concentrations of Fn and Fg in the plasma, this mechanism might result in targeting of S. aureus to fibrin-rich thrombi or elastin-rich tissues.

Interactions of a Periplasmic Binding Protein with a Tetradentate Siderophore Mimic

Iron-bound structure: The ferric complex of a tetradentate siderophore mimic was synthesized and co-crystallized with the periplasmic binding protein CeuE of Campylobacter jejuni. In addition to electrostatic and hydrogen-bonding interactions between the binding pocket and the substrate, the structure showed direct coordination of two amino acid side chains to the Fe(III) center (orange, see figure).

Bacteria in an Intense Competition for Iron: Key Component of the Campylobacter Jejuni Iron Uptake System Scavenges Enterobactin Hydrolysis Product.

Significance Almost all bacteria require Fe(III) for survival and growth. To compete successfully for this essential nutrient, bacteria developed very efficient Fe(III) uptake mechanisms based on high-affinity Fe(III) chelators, so-called siderophores. To gain a competitive advantage, many bacteria have evolved to scavenge and effectively poach siderophores from other species. Enterobactin, one of the strongest Fe(III) chelators known, is produced and secreted by many enteric bacteria. We show that a key protein involved in Fe(III) uptake in the foodborne pathogen Campylobacter jejuni is adapted to scavenge enterobactin hydrolysis products, a strategy that may enable the pathogen to more efficiently exploit siderophores produced by other bacteria and hence their resources. To acquire essential Fe(III), bacteria produce and secrete siderophores with high affinity and selectivity for Fe(III) to mediate its uptake into the cell. Here, we show that the periplasmic binding protein CeuE of Campylobacter jejuni, which was previously thought to bind the Fe(III) complex of the hexadentate siderophore enterobactin (Kd ∼ 0.4 ± 0.1 µM), preferentially binds the Fe(III) complex of the tetradentate enterobactin hydrolysis product bis(2,3-dihydroxybenzoyl-l-Ser) (H5-bisDHBS) (Kd = 10.1 ± 3.8 nM). The protein selects Λ-configured [Fe(bisDHBS)]2− from a pool of diastereomeric Fe(III)-bisDHBS species that includes complexes with metal-to-ligand ratios of 1:1 and 2:3. Cocrystal structures show that, in addition to electrostatic interactions and hydrogen bonding, [Fe(bisDHBS)]2− binds through coordination of His227 and Tyr288 to the iron center. Similar binding is observed for the Fe(III) complex of the bidentate hydrolysis product 2,3-dihydroxybenzoyl-l-Ser, [Fe(monoDHBS)2]3−. The mutation of His227 and Tyr288 to noncoordinating residues (H227L/Y288F) resulted in a substantial loss of affinity for [Fe(bisDHBS)]2− (Kd ∼ 0.5 ± 0.2 µM). These results suggest a previously unidentified role for CeuE within the Fe(III) uptake system of C. jejuni, provide a molecular-level understanding of the underlying binding pocket adaptations, and rationalize reports on the use of enterobactin hydrolysis products by C. jejuni, Vibrio cholerae, and other bacteria with homologous periplasmic binding proteins.

The Three-Dimensional Structure of the Cellobiohydrolase Cel7A from Aspergillus Fumigatus at 1.5 A Resolution

The enzymatic degradation of plant cell-wall cellulose is central to many industrial processes, including second-generation biofuel production. Key players in this deconstruction are the fungal cellobiohydrolases (CBHs), notably those from family GH7 of the carbohydrate-active enzymes (CAZY) database, which are generally known as CBHI enzymes. Here, three-dimensional structures are reported of the Aspergillus fumigatus CBHI Cel7A solved in uncomplexed and disaccharide-bound forms at resolutions of 1.8 and 1.5 Å, respectively. The product complex with a disaccharide in the +1 and +2 subsites adds to the growing three-dimensional insight into this family of industrially relevant biocatalysts.