March 2006
Volume 5
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March 2006
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Contents
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The Slinky as a Ubiquitous Pathogen Recognition Structure
The TLRs are type I integral membrane receptors, each consisting of an N-terminal extracellular PAMP-binding domain, a single transmembrane helix, and a C-terminal, cytoplasmic signaling domain. Our approach was to express large amounts of the extracellular domains (ECDs) of each TLR for X-ray crystallographic analysis and ligand-binding studies. In the paper cited above, we presented the first crystal structure of a TLR ECD, the unliganded form of TLR3-ECD. TLR3 responds to dsRNA from viruses, and we found that purified TLR3-ECD protein binds pI:pC (a dsRNA surrogate) in solution. The TLR3-ECD consists of 23 tandem repeats of a motif known as the leucine-rich repeat (LRR). In three-dimensions, each LRR forms a loop, with consensus hydrophobic residues pointing inward, forming a stabilizing hydrophobic core (Figure 1, part A). When hooked together, the LRRs create a large solenoid in the shape of a horseshoe; overall, the TLR3-ECD can be described as a 23 turn “slinky” (Figure 1, part B). The concave inner surface consists of a large parallel β-sheet, with each β-strand roughly perpendicular to the solenoid axis and linked to the next strand by an irregular loop. LRR12 and LRR20 contain large insertions (Figure 1, parts B and C, highlighted in red). Since these insertions are unique to TLR3 and are conserved in all known mammalian TLR3 orthologs, they likely play important roles in TLR3 function, perhaps in ligand binding. The molecular surface of the TLR3-ECD is abundantly and unevenly populated with N-linked carbohydrates. However, one surface of the ECD is devoid of carbohydrate and free to interact with either ligand or another protein molecule (Figure 1, part C). In the absence of a TLR3-dsRNA complex structure, we can only speculate where ligand binding occurs. However, the presence of bound sulfate molecules from the crystallization medium (Figure 1, parts B and C) provides clues. The sulfate ions mimic the phosphate residues from the nucleotide backbone of a dsRNA molecule, indicating areas of the receptor that are capable of recognizing ligand. Figure 1. Structure of the Toll-like receptor 3 (TLR3) extracellular domain (ECD) and model of the full-length receptor. A) A single leucine-rich repeat (LRR) loop highlighting conserved hydrophobic side chains (brown spheres) that form the core of the solenoid. B) A cartoon trace showing the curved solenoid, or “slinky” shape of the ECD. β-strands are shown as arrows on the concave surface of the ECD. C) A surface rendering of TLR3. In B and C, glycans are shown in green, sulfate ions in orange, and insertions in LRRs 12 and 20 in red. Transmembrane and cytoplasmic domains, based on previously reported structures, are shown in gray. The binding of PAMPs by TLRs triggers inflammatory processes that can have either beneficial or detrimental consequences. Understanding how the recognition of pathogens by TLRs occurs should aid in the development of TLR agonists and antagonists for use as adjuvants in vaccine development, or as anti-inflammatory drugs. This project is a collaboration between the laboratories of David Segal, PhD, Experimental Immunology Branch/National Cancer Institute (NCI), and David Davies, PhD, Laboratory of Molecular Biology/National Institute of Diabetes & Digestive & Kidney Diseases (NIDDK), with help from an intramural biodefense award from the National Institute of Allergy and Infectious Diseases (NIAID).
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considering antigen recognition, antibodies and T-cell receptors, the receptors
of the adaptive immune system, typically come to mind. However, immunologists
have known for some time that other, innate forms of antigen recognition must
exist, since infectious agents are held in check prior to the development of
adaptive immune responses. The most dramatic demonstration of the innate response
is the ability of immunodeficient mice that lack antibodies or T-cell receptors
to survive in non-sterile environments. In both immunodeficient and normal mice,
pathogen invasion results in the immediate recruitment of phagocytes and other
immune cells that ingest the pathogen, produce toxic substances that kill it,
or both. So how, in the absence of T cells and antibodies, are these pathogens
recognized? Recently, a family of homologous proteins known as the Toll-like
receptors (TLRs) was shown to serve just such a pathogen-recognition function.
The TLRs were discovered as homologs of the Drosophila receptor Toll,
an essential component of the immune response to fungi in flies, and it is now
known that similar molecules serve immune functions throughout the animal and
plant kingdoms. In humans, 10 TLR paralogs recognize a wide variety of “pathogen-associated
molecular patterns” (PAMPs), including lipids, proteins, carbohydrates,
and nucleic acids from bacteria, parasites, and viruses. We asked how only 10
germ-line–encoded molecules are able to recognize such a wide variety of
structures at the molecular level.