Summary
Toll-like receptor (TLR) ligand-binding domains comprise 18–25 tandem copies of a 24-residue motif known as the leucine-rich repeat (LRR). Unlike other LRR proteins, TLRs contain significant numbers of non-consensus LRR sequences, which makes their identification by computer domain search programs problematic. Here, we provide methods for identifying non-consensus LRRs. Using the location of these LRRs, hypothetical models are constructed based on the known molecular structures of homologous LRR proteins. However, when a hypothetical model for TLR3 is compared with the molecular structure solved by x-ray crystallography, the solenoid curvature, planarity, and conformations of the LRR insertions are incorrectly predicted. These differences illustrate how non-consensus LRR motifs influence TLR structure. Since the determination of molecular structures by crystallography requires substantial amounts of protein, we describe methods for producing milligram amounts of TLR3 extracellular domain (ECD) protein. The recombinant TLR3-ECD previously used to solve the molecular structure of TLR3-ECD has also been used to study the binding of TLR3-ECD to its ligand, double-stranded RNA (dsRNA). In the last section, we describe the preparation of defined TLR3 ligands and present methods for characterizing their interaction with TLR3-ECD.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Gay, N.J. and Gangloff, M. (2007) Structure and function of toll receptors and their ligands. Annu. Rev. Biochem. 76, 141–165.
Kobe, B. and Kajava, A.V. (2001) The leucine-rich repeat as a protein recognition motif. Curr. Opin. Struct. Biol. 11, 725–732.
Bell, J.K., Mullen, G.E.D., Leifer, C.A., Mazzoni, A., Davies, D.R., and Segal, D.M. (2003) Leucine-rich repeats and pathogen recognition in Toll-like receptors. Trends Immunol. 24, 528–533.
Bell, J.K., Botos, I., Hall, P.R., Askins, J., Shiloach, J., Segal, D.M.,et al. (2005) The molecular structure of the Toll-like receptor 3 ligand-binding domain. Proc. Natl. Acad. Sci. U.S.A. 102, 10976–10980.
Choe, J., Kelker, M.S., and Wilson, I.A. (2005) Crystal structure of human Toll-like receptor 3 (TLR3) ectodomain. Science 309, 581–585.
He, X.L., Bazan, J.F., McDermott, G., Park, J.B., Wang, K., Tessier-Lavigne, M., et al (2003) Structure of the Nogo receptor ectodomain: a recognition module implicated in myelin inhibition. Neuron 38, 177–185.
Finn, R.D., Mistry, J., Schuster-Böckler, B., Griffiths-Jones, S., Hollich, V., Lassmann, T., et al (2006) Pfam: clans, web tools and services. Nucleic Acids Res 34, D247–D251.
Rallabhandi, P., Bell, J., Boukhvalova, M.S., Medvedev, A., Lorenz, E., Arditi, M. et al (2006) Analysis of TLR4 polymorphic variants: new insights into TLR4/MD-2/CD14 stoichiometry, structure, and signaling. J. Immunol. 177, 322–332.
Acknowledgements
This work was supported by the Intramural Research Program of the NIH (NCI and NIDDK) and by a Trans-NIH/FDA Intramural Biodefense Award from NIAID.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science + Business Media, LLC
About this protocol
Cite this protocol
Leonard, J.N., Bell, J.K., Segal, D.M. (2009). Predicting Toll-Like Receptor Structures and Characterizing Ligand Binding. In: McCoy, C.E., O’Neill, L.A.J. (eds) Toll-Like Receptors. Methods in Molecular Biology™, vol 517. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-541-1_4
Download citation
DOI: https://doi.org/10.1007/978-1-59745-541-1_4
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-934115-72-5
Online ISBN: 978-1-59745-541-1
eBook Packages: Springer Protocols