PI: Eklund (SLU)
Project: Ribonucleotide reductase holoenzyme complexes
Structure solution of FimC-FimH complex of E.coli
One of the most important events during the infection by Gram-negative bacteria is their attachment to host cell receptors. In uropathogenic E.coli strains this event is mediated by adhesive surface organelles called Type-1 pili or fimbriae. The fimbriae are large filaments made up of multimeric proteins anchored to the bacterial outer membrane.
The bulk of the pilin filament is made up of a protein called FimA. Associated with FimA, are other proteins like FimG,FimH, FimF etc. FimH binds to mannose and is thought to be responsible for the recognition of specific mannose containing receptors in the surface of the urinary tract cells of the host.
The pilus proteins require the mediation of specific chaperones for their transport across the periplasmic space in E.coli. For the fimbrial proteins, this chaperone is called FimC which is a soluble protein of 205 amino acids.
We have earlier determined the structure of FimC by X-ray diffraction. It was found to consist of two immunoglobulin domains attached by means of a flexible linker. Now we have also determined the structure of the complex of FimC and FimH using X-ray diffraction and Multiple Wavelength Anomalous Dispersion methodology. Like FimC, FimH is also a two domain protein, only one of which is found to be in tight interaction with FimC. At present we have just completed building the initial model of the protein and are about to initiate the refinement process.
Docking of Nucleoside Substrates to Thymidylate kinase (TK)
Thymidylate kinase is responsible of the phosphorylation of nucleosides to make the corresponding nucleotides. Over the years this enzyme has been proposed to be an excellent target for anti-viral chemotherapy. The enzyme has poor substrate specificity. One of the isoforms of the human enzyme called TK2 converts [[alpha]]-D, [[alpha]]-L, [[beta]]-D or [[beta]]-L thymidines to the corresponding monophosphates. In order to understand the mechanism of the apparently unusual promiscuity in substrate utilisation we undertook computational simulations of the substrate binding to this enzyme. The model system used for the study was the Herpes Virus Thymidylate Kinase, of which a crystal structure is already available. The results show that all these substrates take up specific conformational states which result in very similar binding mode within the active site. The conformational differences among the substrates are solely due to their attempt to present the same functional groups to the same residues in the active site, in spite of their major configurational differences. The studies were extended to the 2,3 dideoxy analogues of the substrates which also are recognised by TK. Even these compounds followed the same principles of binding as their parent compounds.
Kerstin Otte, Devapriya Choudhury, Marika Charalambous, Wilhelm Engstrom and Bjorn Rozell (1998). A conserved structural element in horse and mouse IGF2 genes binds a methylation sensitive factor. Nucleic Acids Research, 1998, 26, 1605-1612.