Predicting toll-like receptor structures and characterizing ligand binding

Joshua N. Leonard*, Jessica K. Bell, David M. Segal

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapter

6 Scopus citations

Abstract

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.

Original languageEnglish (US)
Title of host publicationToll-Like Receptors
Subtitle of host publicationMethods and Protocols
EditorsClaire McCoy, Luke O'Neill
Pages55-67
Number of pages13
DOIs
StatePublished - Dec 1 2009

Publication series

NameMethods in Molecular Biology
Volume517
ISSN (Print)1064-3745

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Keywords

  • Double-stranded RNA
  • Homology-based modelling
  • Receptor specificity
  • TLR
  • Toll-like receptor
  • dsRNA

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Leonard, J. N., Bell, J. K., & Segal, D. M. (2009). Predicting toll-like receptor structures and characterizing ligand binding. In C. McCoy, & L. O'Neill (Eds.), Toll-Like Receptors: Methods and Protocols (pp. 55-67). (Methods in Molecular Biology; Vol. 517). https://doi.org/10.1007/978-1-59745-541-1_4