title: Recombinant production of functional full-length and truncated human TRAM/TICAM-2 adaptor protein involved in Toll-like receptor and interferon signaling.
abstract: TRAM/TICAM-2 is used by Toll-like receptor 4 (TLR4) as a bridging adaptor during the mammalian innate immune response. It recruits TRIF, another TIR domain-containing adaptor protein, to TLR4 via TIR domain interactions, which leads to the activation of transcription factors responsible for the production of type-1 interferon and cytokines. The molecular mechanisms of these dual interactions mediated by the TRAM TIR domain are not clear. To understand the molecular basis of TIR:TIR domain interactions, structural and biochemical studies of TRAM TIR domain are necessary, and require a functional soluble protein. In this paper, we report a successful purification and characterization of full-length TRAM. Because full-length TRAM likely contains unstructured regions that may be disadvantageous for structural studies, we also carried out a systematic construct design to determine the boundaries of the TRAM TIR domain. The truncated TRAM constructs were designed based on secondary structure predictions and screened by small-scale expression. Selected constructs were subjected to biophysical analyses. We show that the expressed TRAM TIR domain is functional using in vitro GST pull-down assays that demonstrate a physical interaction with the TLR4 TIR domain. We further show, by site-directed mutagenesis, that the "BB loop" regions of both the TRAM TIR domain and the TLR4 TIR domain are crucial for this physical interaction.
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia; Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia; Australian Infectious Diseases Research Centre, University of Queensland, Brisbane, Queensland 4072, Australia. Electronic address: obayed.ullah@uqconnect.edu.au.
authors.forename: M Obayed
authors.initials: MO
authors.lastname: Ullah
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia; Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia; Australian Infectious Diseases Research Centre, University of Queensland, Brisbane, Queensland 4072, Australia; MRC Laboratory of Molecular Biology, University of Cambridge, Cambridge CB2 0QH, United Kingdom.
authors.forename: Eugene
authors.initials: E
authors.lastname: Valkov
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia; Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia; Australian Infectious Diseases Research Centre, University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Thomas
authors.initials: T
authors.lastname: Ve
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia; Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia; Australian Infectious Diseases Research Centre, University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Simon
authors.initials: S
authors.lastname: Williams
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia; Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia; Centre for Advanced Imaging, The University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Caroline
authors.initials: C
authors.lastname: Mas
authors.affiliation: Centre for Innate Immunity and Infectious Diseases, Monash Institute of Medical Research, Monash University, Melbourne, Victoria 3168, Australia.
authors.forename: Ashley
authors.initials: A
authors.lastname: Mansell
authors.affiliation: School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland 4072, Australia; Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland 4072, Australia; Australian Infectious Diseases Research Centre, University of Queensland, Brisbane, Queensland 4072, Australia. Electronic address: b.kobe@uq.edu.au.
authors.forename: Bostjan
authors.initials: B
authors.lastname: Kobe
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001671:Biochemistry / Q000379:methods*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007372:Interferons / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D066244:Proton Magnetic Resonance Spectroscopy; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction; D051197:Toll-Like Receptor 4 / Q000737:chemistry / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D011994:Recombinant Proteins; C480228:TICAM2 protein, human; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D007372:Interferons
keywords: GST pull-down assays; Innate immunity; Protein expression; Protein purification; Systematic construct design; Toll-like receptor adaptor protein