title: Crystallization and X-ray diffraction analysis of the N-terminal domain of the Toll-like receptor signalling adaptor protein TRIF/TICAM-1.
abstract: As part of the mammalian innate immune response, Toll-like receptors 3 and 4 can signal via the adaptor protein TRIF/TICAM-1 to elicit the production of type-I interferons and cytokines. Recent studies have suggested an auto-inhibitory role for the N-terminal domain (NTD) of TRIF. This domain has no significant sequence similarity to proteins of known structure. In this paper, the crystallization and X-ray diffraction analysis of TRIF-NTD and its selenomethionine-labelled mutant TRIF-NTD(A66M/L113M) are reported. Thin plate-like crystals of native TRIF-NTD obtained using polyethylene glycol 3350 as precipitant diffracted X-rays to 1.9 Å resolution. To facilitate phase determination, two additional methionines were incorporated into the protein at positions chosen based on the occurrence of methionines in TRIF homologues in different species. Crystals of the selenomethionine-labelled protein were obtained under conditions similar to the wild-type protein; these crystals diffracted X-rays to 2.5 Å resolution. The TRIF-NTD and TRIF-NTD(A66M/L113M) crystals have the symmetry of space groups P212121 and P1, and most likely contain two and four molecules in the asymmetric unit, respectively. These results provide a sound foundation for the future structure determination of this novel domain.
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: M Obayed
authors.initials: MO
authors.lastname: Ullah
authors.forename: Thomas
authors.initials: T
authors.lastname: Ve
authors.forename: Jameris
authors.initials: J
authors.lastname: Dkhar
authors.forename: Mohammed
authors.initials: M
authors.lastname: Alaidarous
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Ericsson
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Sweet
authors.forename: Ashley
authors.initials: A
authors.lastname: Mansell
authors.forename: Bostjan
authors.initials: B
authors.lastname: Kobe
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003460:Crystallization; D018360:Crystallography, X-Ray; D006801:Humans; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012645:Selenomethionine / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D050505:Mutant Proteins; C470409:TICAM1 protein, human; D012645:Selenomethionine
keywords: Toll-like receptors; adaptor proteins; innate immunity; introduction of additional methionine residues; selenomethionine labelling