TY - JOUR
T1 - Recombinant production of functional full-length and truncated human TRAM/TICAM-2 adaptor protein involved in Toll-like receptor and interferon signaling
AU - Ullah, M. Obayed
AU - Valkov, Eugene
AU - Ve, Thomas
AU - Williams, Simon
AU - Mas, Caroline
AU - Mansell, Ashley
AU - Kobe, Bostjan
N1 - Publisher Copyright:
© 2014 Elsevier Inc. All rights reserved.
PY - 2015/2
Y1 - 2015/2
N2 - 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.
AB - 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.
KW - GST pull-down assays
KW - Innate immunity
KW - Protein expression
KW - Protein purification
KW - Systematic construct design
KW - Toll-like receptor adaptor protein
UR - http://www.scopus.com/inward/record.url?scp=84911456248&partnerID=8YFLogxK
U2 - 10.1016/j.pep.2014.09.019
DO - 10.1016/j.pep.2014.09.019
M3 - Article
SN - 1046-5928
VL - 106
SP - 31
EP - 40
JO - Protein Expression and Purification
JF - Protein Expression and Purification
ER -