Position: "Forskare" (SLU)
Project: Structure-function studies of virulence-associated
adhesion organelles from pathogenic Gram negative bacteria.
Funding ends: 31 December, 2000
Based on our structure of the FimC-FimH chaperone-adhesin complex (published in Science 1999), we had previously identified a potential mono-mannose binding pocket at the tip of the mannose-binding lectin domain of FimH. Using a cavity-finding algorithm developed by Devapriya Choudhury we have now identified a number of potential additional carbohydrate-binding sites distributed over the surface of the FimH lectin domain. Variations in FimH binding profiles due to allelic variation or random mutagenesis correlate with these cavities. Docking studies using Autodock show that all of the identified cavities are able to accommodate up to tri-saccharide units. The emerging picture is that there are two main regions of FimH-carbohydrate recognition; a tip-located surface mediating tight binding to high-mannose receptors, and a second surface on the side of the molecule with less specificity and lower affinity which may modulate binding affinity as well as confer capability to bind e.g. complex oligosaccharides. Mutational and structural studies to further dissect the structural details of FimH-carbohydrate interactions are under way.
During the year, I have established a collaboration with Dr. Sheila MacIntyre (University of Reading, UK) and Dr. Vladimir Zav'yalov (Institute of Immunological Engineering, Moscow Region, Russia) aimed at elucidating the structure and assembly of the F1 capsular antigen from Yersinia pesits. This organelle is the prototype for a class of thin fibrillar adhesive adhesins (TAFA) assembled via the FGL-type chaperone/usher system, for which there is currently no structural information available. We have recently been able to show that the F1 antigen is a linear array of Caf1 subunits joined head-to-tail by a donor strand complementation mechanism similar to that used for pilus assembly. A stable and soluble Caf1M-Caf1 complex suitable for X-ray crystallography has been constructed, expressed, and purified. A post-doc to work on the structure determination of this complex has been appointed, although the current funding situation is problematic and only allows for a three-month stay.
The structure determination of the FimC-FimH complex has continued to generate interest both internationally and at home, with three invited reviews and six invited lectures during the year.
A personal highlight of the year was that I finally managed to get a real job by being appointed as "lektor" in biochemistry at SLU.
Sources of funding 2000
SBNet (1 Ph.D. position, 1 researcher position)
Publications 2000
1. Knight SD (2000): RSPS version 4: a semi-interactive
vector-search program for solving heavy-atom derivatives.
Acta Cryst, D56:42-47.
2. Sauer FG, Knight SD, Waksman G & Hultgren SJ
(2000): PapD-like chaperones and pilus biogenesis.
Sem. Cell. Dev. Biol., 11:27-34.
3. Sauer FG, Barnhart M, Choudhury D, Knight SD, Waksman
G & Hultgren SJ (2000): Chaperone-assisted pilus
assembly and bacterial attachment. Curr. Opin. Struc.
Biol. 10(5):548-556.
4. Knight SD, Berglund J & Choudhury D (2000): Bacterial
adhesins: structural studies reveal chaperone function
and pilus biogenesis. Curr. Opin. Chem. Biol. 4(6):653-660.
Invited lectures 2000
* Donor Strand Complementation in Pilus Biogenesis.
BioInvent (host: Eskil Söderlind), Lund, Sweden,
2000.
* Donor Strand Complementation in Pilus Biogenesis.
Gordon Research Conference on Reversible Associations
in Structural and Molecular Biology, Ventura, California,
USA, 2000.
* Donor Strand Complementation in Pilus Biogenesis.
Department of Medical Biochemistry and Biophysics,
Karolinska Institute (host: Gunter Schneider), Stockholm,
Sweden, 2000.
* Donor Strand Complementation in Pilus Biogenesis.
EMBL Conference: Millennium Symposium on Structural
Biology, Heidelberg, Germany, 2000.
* Steric chaperones in assembly of bacterial adhesins.
Umeå University (host: Uwe Sauer), Umeå,
Sweden, 2000.
* Structural Basis for Bacterial Recognition and Binding
in the Urinary Tract. Colloquium on Protein-Carbohydrate
interactions II, Vrije Universiteit Brussel, Brussels,
Belgium, 2000.
Ongoing scientific collaborations
* Dr. Colin Hughes, University of Cambridge, UK (Flagellar
assembly)
* Dr. Scott Hultgren, Washington University School of
Medicine, St Louis, USA (Type 1 pili)
* Dr. Per Klemm, Technical University of Denmark, Lyngby,
Denmark (Bacterial adhesins)
* Dr. Sheila MacIntyre, University of Reading, UK (F1
antigen)
* Dr. Hans Wulf-Watz, Umeå University, Sweden
(Type III secretion LcrV pore forming protein)
* Dr. Vladimir Zav'yalov, Institute of Immunological
Engineering, Moscow Region, Russia (F1 antigen)
Latest update at 5 March, 2001.