Open AccessCrystallization and X‐ray diffraction analysis of the N‐terminal domain of the Toll‐like receptor signalling adaptor protein TRIF/TICAM‐1
Author(s) -
Ullah M. Obayed,
Ve Thomas,
Dkhar Jameris,
Alaidarous Mohammed,
Ericsson Daniel J.,
Sweet Matthew J.,
Mansell Ashley,
Kobe Bostjan
Publication year - 2013
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s174430911301419x
Subject(s) - trif , signal transducing adaptor protein , receptor , chemistry , innate immune system , toll like receptor , biochemistry
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 P 2 1 2 1 2 1 and P 1, 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.