Toll like receptors (TLRs) are the signaling component of the innate immune system in mammals, which constitutes the first line of defense against microbial infection and endogenous danger signals. They recognize the invading pathogens during early stages of infection and initiate signaling for immune responses such as proinflammatory cytokine production. To signal down-stream, activated TLRs recruit one or several cytosolic adaptors. The recruitment of specific adaptor proteins determines the specificity of inflammatory response, via the activation of distinct transcription factors and pro-inflammatory genes. TRIF (TIR containing adaptor inducing IFN-β) and TRAM (TRIF related adaptor molecule) are involved in the signaling pathways of TLR3 and TLR4. They are TIR (Toll/interleukin-1 receptor) domain-containing adaptors in the cytosol and are involved in down-stream signaling directly via their conserved TIR domain. TRIF can interact alone with the TIR domain of TLR3. TRAM is required for the interaction of TRIF with the TIR domain of TLR4. The mechanism of the assembly of a TLR with these adaptor proteins and the structural basis of the interactions of different domains of TRIF and TRAM are not clearly understood. In this study, I aimed to determine the three-dimensional structure of different domains of TRIF and TRAM to understand the molecular basis of interaction of TRIF and TRAM in TLR signaling complexes.
In the first part of the thesis, I present the structural characterization of the N-terminal domain of TRIF (TRIF-NTD). Chapter 2 and Chapter 3 of the thesis describe the details of the crystallization, structure determination and functional characterization of TRIF-NTD. In Chapter 2, I describe the screening of TRIF-NTD constructs to identify suitable constructs for crystallization. The expression and purification of TRIF-NTD and crystallization of this domain are also described in this chapter. Chapter 3 contains the details of structure determination and functional characterization of TRIF-NTD. Structural analysis reveals that TRIF-NTD has a novel fold containing 8 anti-parallel helices, sharing similarities with the interferon-induced protein with tetratricopeptide repeats (IFIT) family of proteins, which are involved in both recognition of viral RNA and modulation of innate immune signaling. Analysis of TRIF-NTD surface features and mapping of sequence conservation onto the structure suggest several possible binding sites involved in either TRIF auto-regulation or the interaction with other signaling molecules or ligands. In agreement to previous reports, TRIF-NTD suppressed TRIF-mediated activation of the interferon-β promoter, as well as NF-κB-dependent reporter gene activity.
The second part of the thesis describes the details of the expression, purification, characterization and crystallization of the TIR domains of TRIF and TRAM. In Chapter 4, I describe the production and purification of TRIF TIR domain. From the large-scale expression, it was concluded that TRIF TIR domain constructs are expressed, but none of them produce soluble protein in E. coli. In Chapter 5, I elaborate on the details of the production, purification and characterization of TRAM protein. From high-throughput screening of different constructs of TRAM in E. coli, I identified the constructs that produce soluble protein. From the solubility profile of different constructs of TRAM, I identified the boundaries of the TRAM TIR domain. I also describe the large-scale expression, purification and characterization of different TRAM constructs in Chapter 5, which identified the best TRAM construct for structural studies. Chapter 6 includes the details of the efforts made to crystalize the TRAM TIR domain. Several approaches including thermofluor-based buffer optimization, surface entropy reduction, MBP (maltose binding protein)-driven crystallization and metal-mediated crystallization were attempted in order to crystalize the TRAM TIR domain.
In summary, this thesis has made significant contributions towards the understanding of the molecular mechanisms of the assembly of TLR signaling complexes. In particular, it has taken us one step closer to understanding the molecular basis of the interactions of TRIF in TLR signaling complexes and the auto-regulation of TRIF and thereby identified opportunities for selective targeting of TLR3 and TLR4-mediated inflammation.