Biomedical subjects

Wolfgang Wende

Publications and source records attributed to Wolfgang Wende.

6 recordsLinked to original sources

PubMed2006

From monomeric to homodimeric endonucleases and back: engineering novel specificity of LAGLIDADG enzymes.

Monomeric homing endonucleases of the LAGLIDADG family recognize DNA in a bipartite manner, reflecting the underlying structural assembly of two protein domains (A and B) related by pseudo 2-fold symmetry. This architecture allows for changes in DNA specificity via the distinct combination of these half-site domains. The key to engineering such hybrid proteins lies in the LAGLIDADG two-helix bundle that forms both the domain interface and the endonuclease active site. In this study, we utilize domain A of the monomeric I-DmoI to demonstrate the feasibility of generating functional homodimeric endonucleases that recognize palindromic DNA sequences derived from the original, non-palindromic target. Wild-type I-DmoI domain A is capable of forming a homodimer (H-DmoA) that binds tightly to, but does not cleave efficiently, its anticipated DNA target. Partial restoration of DNA cleavage ability was obtained by re-engineering the LAGLIDADG dimerization interface (H-DmoC). Upon fusing two copies of H-DmoC via a short peptide linker, a novel, site-specific DNA endonuclease was created (H-DmoC2). Like I-DmoI, H-DmoC2 is thermostable and cleaves the new target DNA to generate the predicted 4 nt 3'-OH overhangs but, unlike I-DmoI, H-DmoC2 retains stringent cleavage specificity when substituting Mn2+ for Mg2+ as co-factor. This novel endonuclease allows speculation regarding specificity of monomeric LAGLIDADG proteins, while it supports the evolutionary genesis of these proteins by a gene duplication event.

Amino Acid Motifs↗

PubMed2005

Plasmodium falciparum glutathione S-transferase--structural and mechanistic studies on ligand binding and enzyme inhibition.

Glutathione S-transferase of the malarial parasite Plasmodium falciparum (PfGST) represents a novel class of GST isoenzymes. Since the architecture of the PfGST substrate binding site differs significantly from its human counterparts and there is only this one isoenzyme present in the parasite, PfGST is considered a highly attractive target for antimalarial drug development. Here we report the mechanistic, kinetic, and structural characterization of PfGST as well as its interaction with different ligands. Our data indicate that in solution PfGST is present as a tetramer that dissociates into dimers in the presence of glutathione (GSH). Fluorescence spectroscopy shows that in the presence of GSH GST serves as ligandin for parasitotoxic ferriprotoporphyrin IX with a high- and a low-affinity binding site. This is supported by a clear uncompetitive inhibition type. Site-directed mutagenesis studies demonstrate that neither Cys 86 nor Cys 101 contribute to the peroxidase activity of the enzyme, which is thus performed GSH-dependently at the active site. Tyr 9 is responsible for the deprotonation of GSH and Lys 15, but also Gln 71 are involved in GSH binding. We furthermore report the 2.4 A resolution X-ray structure of PfGST cocrystallized with the inhibitor S-hexylglutathione. In comparison with a previously reported structure obtained by crystal soaking, differences occur at the C-terminal end of helix alpha4 and at the S-hexylmoiety of the inhibitor. We furthermore show that, in contrast to previous reports, the antimalarial drug artemisinin is not metabolized by PfGST.

Animals↗

PubMed2005

Developing a programmed restriction endonuclease for highly specific DNA cleavage.

Specific cleavage of large DNA molecules at few sites, necessary for the analysis of genomic DNA or for targeting individual genes in complex genomes, requires endonucleases of extremely high specificity. Restriction endonucleases (REase) that recognize DNA sequences of 4-8 bp are not sufficiently specific for this purpose. In principle, the specificity of REases can be extended by fusion to sequence recognition modules, e.g. specific DNA-binding domains or triple-helix forming oligonucleotides (TFO). We have chosen to extend the specificity of REases using TFOs, given the combinatorial flexibility this fusion offers in addressing a short, yet precisely recognized restriction site next to a defined triple-helix forming site (TFS). We demonstrate here that the single chain variant of PvuII (scPvuII) covalently coupled via the bifunctional cross-linker N-(gamma-maleimidobutryloxy) succinimide ester to a TFO (5'-NH2-[CH2](6 or 12)-MPMPMPMPMPPPPPPT-3', with M being 5-methyl-2'-deoxycytidine and P being 5-[1-propynyl]-2'-deoxyuridine), cleaves DNA specifically at the recognition site of PvuII (CAGCTG) if located in a distance of approximately one helical turn to a TFS (underlined) complementary to the TFO ('addressed' site: 5'-TTTTTTTCTCTCTCTCN(approximately 10)CAGCTG-3'), leaving 'unaddressed' PvuII sites intact. The preference for cleavage of an 'addressed' compared to an 'unaddressed' site is >1000-fold, if the cleavage reaction is initiated by addition of Mg2+ ions after preincubation of scPvuII-TFO and substrate in the absence of Mg2+ ions to allow triple-helix formation before DNA cleavage. Single base pair substitutions in the TFS prevent addressed DNA cleavage by scPvuII-TFO.

Cross-Linking Reagents↗

PubMed2004

Chimeras of the homing endonuclease PI-SceI and the homologous Candida tropicalis intein: a study to explore the possibility of exchanging DNA-binding modules to obtain highly specific endonucleases with altered specificity.

Homing endonucleases are extremely specific endodeoxyribonucleases. In vivo, these enzymes confer mobility on their genes by inducing a very specific double-strand cut in cognate alleles that lack the cooling sequence for the homing endonuclease; the cellular repair of the double-strand break with the endonuclease-containing allele as a template leads to integration of the endonuclease gene, completing the homing process. As a result of their extreme sequence specificity, homing endonucleases are promising tools for genome engineering. For this purpose, it is desirable to design enzymes with defined new specificities. To analyse which DNA-binding elements are potential candidates for use in the design of enzymes with modified or even new specificity, we produced several chimeric proteins derived from the Saccharomyces cerevisiae VMA1 intein (PI-SceI) and the related Candida tropicalis VMA1 intein. Although the mature Candida intein is devoid of endonucleolytic activity, the exchange of two DNA-binding modules of PI-SceI with the homologous elements from the Candida intein results in an active endonuclease. The low sequence homology in these modules indicates that different protein-DNA contacts are responsible for the recognition of related DNA sequences. This flexibility in DNA recognition should, in principle, allow endonucleases to be produced with new specificities useful for genome engineering.

Amino Acid Sequence↗

PubMed2003

DNA recognition by the homing endonuclease PI-SceI involves a divalent metal ion cofactor-induced conformational change.

PI-SceI, a homing endonuclease of the LAGLIDADG family, consists of two domains involved in DNA cleavage and protein splicing, respectively. Both domains cooperate in binding the recognition sequence. Comparison of the structures of PI-SceI in the absence and presence of substrate reveals major conformational changes in both the protein and DNA. Notably, in the protein-splicing domain the loop comprising residues 53-70 and adopts a "closed" conformation, thus enabling it to interact with the DNA. We have studied the dynamics of DNA binding and subsequent loop movement by fluorescence techniques. Six amino acids in loop53-70 were individually replaced by cysteine and modified by fluorescein. The interaction of the modified PI-SceI variants with the substrate, unlabeled or labeled with tetramethylrhodamine, was analyzed in equilibrium and stopped-flow experiments. A kinetic scheme was established describing the interaction between PI-SceI and DNA. It is noteworthy that the apparent hinge-flap motion of loop53-70 is only observed in the presence of a divalent metal ion cofactor. Substitution of the major Mg2+-binding ligands in PI-SceI, Asp-218 and Asp-326, by Asn or "nicking" PI-SceI with trypsin at Arg-277, which interferes with formation of an active enzyme.substrate complex, both prevent the conformational change of loop53-70. Deletion of the loop inactivates the enzyme. We conclude that loop53-70 is an important structural element that couples DNA recognition by the splicing domain with DNA cleavage by the catalytic domain and as such "communicates" with the Mg2+ binding sites at the catalytic centers.

Aspartic Acid↗

PubMed2002

High resolution crystal structure of domain I of the Saccharomyces cerevisiae homing endonuclease PI-SceI.

The homing endonuclease PI-SceI from Saccharo myces cerevisiae consists of two domains. The protein splicing domain I catalyzes the excision of the mature endonuclease (intein) from a precursor protein and the religation of the flanking amino acid sequences (exteins) to a functional protein. Furthermore, domain I is involved in binding and recognition of the specific DNA substrate. Domain II of PI-SceI, the endonuclease domain, which is structurally homologous to other homing endonucleases from the LAGLIDADG family, harbors the endonucleolytic center of PI-SceI, which in vivo initiates the homing process by introducing a double-strand cut in the approximately 35 bp recognition sequence. At 1.35 A resolution, the crystal structure of PI-SceI domain I provides a detailed view of the part of the protein that is responsible for tight and specific DNA binding. A geometry-based docking of the 75 degrees bent recognition sequence to the full-length protein implies a conformational change or hinge movement of a subdomain of domain I, the tongs part, that is predicted to reach into the major groove near base pairs +16 to +18.

Binding Sites↗