Biochemical and functional analyses of the human Toll-like receptor 3 ectodomain.
The structure of the human Toll-like receptor 3 (TLR3) ectodomain (ECD) was recently solved by x-ray crystallography, leading to a number of models concerning TLR3 function (Choe, J., Kelker, M. S., and Wilson, I. A. (2005) Science 309, 581-585; Bell, J. K., Botos, I., Hall, P. R., Askins, J., Shiloach, J., Segal, D. M., and Davies, D. R. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 10976-10980) The structure revealed four pairs of cysteines that are putatively involved in disulfide bond formation, several residues that are predicted to be involved in dimerization between ECD subunits, and surfaces that could bind to poly(I:C). In addition, there are two loops that protrude from the central solenoid structure of the protein. We examined the recombinant TLR3 ECD for disulfide bond formation, poly(I:C) binding, and protein-protein interaction. We also made over 80 mutations in the residues that could affect these features in the full-length TLR3 and examined their effects in TLR3-mediated NF-kappaB activation. A number of mutations that affected TLR3 activity also affected the ability to act as dominant negative inhibitors of wild type TLR3. Loss of putative RNA binding did not necessarily affect dominant negative activity. All of the results support a model where a dimer of TLR3 is the form that binds RNA and activates signal transduction.
Ranjith-Kumar
C T
CT
Department of Biochemistry and Biophysics, Department of Biology, and Laboratory for Biological Mass Spectrometry, Department of Chemistry, Texas A&M University, College Station, Texas 77843, USA.
Miller
William
W
Xiong
Jin
J
Russell
William K
WK
Lamb
Roberta
R
Santos
Jonathan
J
Duffy
Karen E
KE
Cleveland
Larissa
L
Park
Mary
M
Bhardwaj
Kanchan
K
Wu
Zhaoxiang
Z
Russell
David H
DH
Sarisky
Robert T
RT
Mbow
M Lamine
ML
Kao
C Cheng
CC
D000595:Amino Acid Sequence; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D011070:Poly I-C / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D051196:Toll-Like Receptor 3 / Q000737:chemistry* / Q000502:physiology
D004220:Disulfides; D016328:NF-kappa B; C495344:TLR3 protein, human; D051196:Toll-Like Receptor 3; D011070:Poly I-C