Crystallization and X-ray diffraction analysis of the N-terminal domain of the Toll-like receptor signalling adaptor protein TRIF/TICAM-1

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Ullah, M Obayed
Ve, Thomas
Dkhar, Jameris
Alaidarous, Mohammed
Ericsson, Daniel J
Sweet, Matthew J
Mansell, Ashley
Kobe, Bostjan
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2013
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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 autoinhibitory 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-NTDA66M/L113M are reported. Thin plate-like crystals of native TRIF-NTD obtained using polyethylene glycol 3350 as precipitant diffracted X-rays to 1.9 A˚ 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 A˚ resolution. The TRIF-NTD and TRIF-NTDA66M/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.

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Acta Crystallographica Section F: Structural Biology and Crystallization Communications

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F69

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7

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Biochemistry and cell biology not elsewhere classified

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