Norbert Schormann

Uracil‐DNA glycosylases—Structural and functional perspectives on an essential family of DNA repair enzymes

Uracil‐DNA glycosylases (UDGs) are evolutionarily conserved DNA repair enzymes that initiate the base excision repair pathway and remove uracil from DNA. The UDG superfamily is classified into six families based on their substrate specificity. This review focuses on the family I enzymes since these are the most extensively studied members of the superfamily. The structural basis for substrate specificity and base recognition as well as for DNA binding, nucleotide flipping and catalytic mechanism is discussed in detail. Other topics include the mechanism of lesion search and molecular mimicry through interaction with uracil‐DNA glycosylase inhibitors. The latest studies and findings detailing structure and function in the UDG superfamily are presented.

Crystal structure of vaccinia virus uracil-DNA glycosylase reveals dimeric assembly

BackgroundUracil-DNA glycosylases (UDGs) catalyze excision of uracil from DNA. Vaccinia virus, which is the prototype of poxviruses, encodes a UDG (vvUDG) that is significantly different from the UDGs of other organisms in primary, secondary and tertiary structure and characteristic motifs. It adopted a novel catalysis-independent role in DNA replication that involves interaction with a viral protein, A20, to form the processivity factor. UDG:A20 association is essential for assembling of the processive DNA polymerase complex. The structure of the protein must have provisions for such interactions with A20. This paper provides the first glimpse into the structure of a poxvirus UDG.ResultsResults of dynamic light scattering experiments and native size exclusion chromatography showed that vvUDG is a dimer in solution. The dimeric assembly is also maintained in two crystal forms. The core of vvUDG is reasonably well conserved but the structure contains one additional β-sheet at each terminus. A glycerol molecule is found in the active site of the enzyme in both crystal forms. Interaction of this glycerol molecule with the protein possibly mimics the enzyme-substrate (uracil) interactions.ConclusionThe crystal structures reveal several distinctive features of vvUDG. The new structural features may have evolved for adopting novel functions in the replication machinery of poxviruses. The mode of interaction between the subunits in the dimers suggests a possible model for binding to its partner and the nature of the processivity factor in the polymerase complex.

Structure‐based approach to pharmacophore identification, in silico screening, and three‐dimensional quantitative structure–activity relationship studies for inhibitors of Trypanosoma cruzi dihydrofolate reductase function

We have employed a structure‐based three‐dimensional quantitative structure–activity relationship (3D‐QSAR) approach to predict the biochemical activity for inhibitors of T. cruzi dihydrofolate reductase‐thymidylate synthase (DHFR‐TS). Crystal structures of complexes of the enzyme with eight different inhibitors of the DHFR activity together with the structure in the substrate‐free state (DHFR domain) were used to validate and refine docking poses of ligands that constitute likely active conformations. Structural information from these complexes formed the basis for the structure‐based alignment used as input for the QSAR study. Contrary to indirect ligand‐based approaches the strategy described here employs a direct receptor‐based approach. The goal is to generate a library of selective lead inhibitors for further development as antiparasitic agents. 3D‐QSAR models were obtained for T. cruzi DHFR‐TS (30 inhibitors in learning set) and human DHFR (36 inhibitors in learning set) that show a very good agreement between experimental and predicted enzyme inhibition data. For crossvalidation of the QSAR model(s), we have used the 10% leave‐one‐out method. The derived 3D‐QSAR models were tested against a few selected compounds (a small test set of six inhibitors for each enzyme) with known activity, which were not part of the learning set, and the quality of prediction of the initial 3D‐QSAR models demonstrated that such studies are feasible. Further refinement of the models through integration of additional activity data and optimization of reliable docking poses is expected to lead to an improved predictive ability. Proteins 2008.

Structures of dihydrofolate reductase-thymidylate synthase of Trypanosoma cruzi in the folate-free state and in complex with two antifolate drugs, trimetrexate and methotrexate.

The flagellate protozoan parasite Trypanosoma cruzi is the pathogenic agent of Chagas disease (also called American trypanosomiasis), which causes approximately 50,000 deaths annually. The disease is endemic in South and Central America. The parasite is usually transmitted by a blood-feeding insect vector, but can also be transmitted via blood transfusion. In the chronic form, Chagas disease causes severe damage to the heart and other organs. There is no satisfactory treatment for chronic Chagas disease and no vaccine is available. There is an urgent need for the development of chemotherapeutic agents for the treatment of T. cruzi infection and therefore for the identification of potential drug targets. The dihydrofolate reductase activity of T. cruzi, which is expressed as part of a bifunctional enzyme, dihydrofolate reductase-thymidylate synthase (DHFR-TS), is a potential target for drug development. In order to gain a detailed understanding of the structure-function relationship of T. cruzi DHFR, the three-dimensional structure of this protein in complex with various ligands is being studied. Here, the crystal structures of T. cruzi DHFR-TS with three different compositions of the DHFR domain are reported: the folate-free state, the complex with the lipophilic antifolate trimetrexate (TMQ) and the complex with the classical antifolate methotrexate (MTX). These structures reveal that the enzyme is a homodimer with substantial interactions between the two TS domains of neighboring subunits. In contrast to the enzymes from Cryptosporidium hominis and Plasmodium falciparum, the DHFR and TS active sites of T. cruzi lie on the same side of the monomer. As in other parasitic DHFR-TS proteins, the N-terminal extension of the T. cruzi enzyme is involved in extensive interactions between the two domains. The DHFR active site of the T. cruzi enzyme shows subtle differences compared with its human counterpart. These differences may be exploited for the development of antifolate-based therapeutic agents for the treatment of T. cruzi infection.

Identification of Inhibitors that Block Vaccinia Virus Infection by Targeting the DNA Synthesis Processivity Factor D4

Smallpox was globally eradicated 30 years ago by vaccination. The recent threat of bioterrorism demands the development of improved vaccines and novel therapeutics to effectively preclude a reemergence of smallpox. One new therapeutic target is the vaccinia poxvirus processivity complex, comprising D4 and A20 proteins that enable the viral E9 DNA polymerase to synthesize extended strands. Five compounds identified from an AlphaScreen assay designed to disrupt A20:D4 binding were shown to be effective in: (i) blocking vaccinia processive DNA synthesis in vitro, (ii) preventing cellular infection with minimal cytotoxicity, and (iii) binding to D4, as evidenced by ThermoFluor. The EC(50) values for inhibition of viral infectivity ranged from 9.6 to 23 μM with corresponding selectivity indices (cytotoxicity CC(50)/viral infectivity EC(50)) of 3.9 to 17.8. The five compounds are thus potential therapeutics capable of halting smallpox DNA synthesis and infectivity through disruptive action against a component of the vaccinia processivity complex.

Structures of vaccinia virus dUTPase and its nucleotide complexes.

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate in the presence of Mg(2+) ions. The enzyme plays multiple cellular roles by maintaining a low dUTP:dTTP ratio and by synthesizing the substrate for thymidylate synthase in the biosynthesis of dTTP. Although dUTPase is an essential enzyme and has been established as a valid target for drug design, the high degree of homology of vaccinia virus dUTPase to the human enzyme makes the identification of selective inhibitors difficult. The crystal structure of vaccinia virus dUTPase has been solved and the active site has been mapped by crystallographic analysis of the apo enzyme and of complexes with the substrate-analog dUMPNPP, with the product dUMP and with dUDP, which acts as an inhibitor. Analyses of these structures reveal subtle differences between the viral and human enzymes. In particular, the much larger size of the central channel at the trimer interface suggests new possibilities for structure-based drug design. Vaccinia virus is a prototype of the poxviruses.

Trypanosoma cruzi genome encodes a pteridine reductase 2 protein.

Pteridine metabolism in Trypanosoma cruzi is poorly understood. The term ‘pteridine’ is used collectively for two classes of structurally-related compounds, folates and biopterins, which differ only in the nature of the side chain attached to the C6 atom of the pterin ring. Both folate and biopterin, in their reduced (tetrahydro) forms, serve as essential cofactors in a number of critical metabolic steps in many organisms [1]. While some microorganisms and parasites such as Plasmodium can synthesize folate, mammals and trypanosomatid parasites lack this ability. On the other hand, mammalian cells can synthesize tetrahydrobiopterin de novo from GTP, while these parasites cannot synthesize biopterin either [2–4]. In order to meet the need for these essential nutrients, folate and biopterin are transported from the host into the parasite, and are subsequently reduced to their respective dihydro and tetrahydro forms by parasitic dihydrofolate reductase (DHFR) and pteridine reductase (PTR1) enzymes [1,2]. DHFR is one of the best characterized enzymes. Structure function relationships in DHFR from a variety of sources have been studied in detail and a number of inhibitors targeting DHFR has been successfully used in cancer chemotherapy and against some infectious pathogens including malaria parasite [1,5,6]. On the other hand, much of our current knowledge about pteridine reductase 1 (PTR1) is derived from research in Leishmania [7–12]. The Leishmania gene encoding PTR1 enzyme was found to be responsible for resistance to the classical antifolate drug methotrexate (MTX). The enzyme belongs to the family of short-chain dehydrogenases/reductases (SDR). Biochemical studies showed that it had broad substrate specificity and was able to reduce both folates and biopterins using NADPH as cofactor. PTR1 is considerably less sensitive (at

Identification of Protein-Protein Interaction Inhibitors Targeting Vaccinia Virus Processivity Factor for Development of Antiviral Agents

ABSTRACT Poxvirus uracil DNA glycosylase D4 in association with A20 and the catalytic subunit of DNA polymerase forms the processive polymerase complex. The binding of D4 and A20 is essential for processive polymerase activity. Using an AlphaScreen assay, we identified compounds that inhibit protein-protein interactions between D4 and A20. Effective interaction inhibitors exhibited both antiviral activity and binding to D4. These results suggest that novel antiviral agents that target the protein-protein interactions between D4 and A20 can be developed for the treatment of infections with poxviruses, including smallpox.

Structural genomics of caenorhabditis elegans: Crystal structure of calmodulin

Introduction. Calmodulin (CaM), a conserved eucaryotic protein, can bind specifically to a large number of intracellular proteins and modulate their activity in response to the Ca concentration. This small 17-kDa acidic protein belongs to a family of homologous calcium-binding proteins that bind Ca through the EF-hand motif (e.g., parvalbumin or troponin C). A compact, calcium-free, apo form of CaM is converted to an extended dumbbellshaped form on binding Ca . The extended conformation of CaM has been by far the most thoroughly studied, especially by X-ray crystallography. It consists of two structurally similar domains separated by a flexible 28-residue helix. Each domain has two EF-hand motifs with bound Ca . The calciuminduced extension of CaM exposes two hydrophobic pockets, one per domain, which represent the binding sites for target proteins. In some protein targets, the CaM-binding region was located to a sequence of 18 amino acids predicted to form an -helix. On binding to the protein target, the central CaM helix unwinds, and the two hydrophobic pockets wrap around the -helix of the protein target. Structural plasticity of the hydrophobic pockets and flexibility of the central helix are thought to account for the ability of CaM to interact with a variety of different targets in a sequence-independent fashion. We have determined the crystal structure of calciumbound CaM from Caenorhabditis elegans (ceCaM) as a part of the Structural Genomics of C. elegans project. Besides the conserved features typical for all CaM’s, the ceCaM structure has the straightest central helix so far observed in CaM’s. This relatively straight helix may be induced by different crystallization conditions and/or by the crystal symmetry.

Structure of the uracil complex of Vaccinia virus uracil DNA glycosylase

The crystal structure of the uracil complex of Vaccinia virus uracil DNA glycosylase (D4) has been determined at 2.03 Å resolution.