Mapping subunit organisation within the T-Cell Receptor-CD3 complex

© 2017 Dr. Logesvaran KrshnanIn the mammalian immune system, T cells play important roles in mounting an effective and sustained immune response for protection against infection. One of the major molecules that contributes to T cell development and function is the cell surface receptor, T cell receptor (TCR). TCR recognises an antigenic peptide fragment presented by a major histocompatibility (MHC) molecule on antigen-presenting cells and subsequently initiates intracellular signalling. The T cell receptor complex is composed of ligand-sensing subunits (TCRα and TCRβ chains) and signal-transducing subunits (CD3δ, CD3γ, CD3ε and ζ chains). To transmit the extracellular ligand binding to cell interior, TCR chains communicate with the signal-transducing subunits of the receptor complex through unknown mechanisms. How subunits of the TCR-CD3 complex (hereafter referred to as the TCR complex) communicate with each other is unclear largely due to an absence of information regarding the molecular architecture of the intact TCR complex. In this thesis, I aimed to map subunit organisation within the TCR complex based on their transmembrane (TM) domain arrangement. Results presented in this thesis highlight the successful use of a cysteine crosslink screening approach to obtain TM-TM relationships within the TCR complex. I incorporated the crosslinking data along with biophysical, computational and functional methods to interrogate the architecture of the TCR complex and possible features of the TCR TM domains in mediating TCR triggering events. In chapter 3, I demonstrated that the TCRα and TCRβ subunits are closely associated in the membrane using cysteine crosslinking data and MD simulation-generated TM structures of the TCRαβ heterodimer. I described a novel polar network within the TCRαβ TM interface and demonstrated by mutagenesis that association of the TCRαβ TM domains through this polar network is crucial to maintain native TCR complex assembly and surface expression. In chapter 4, I have extended the cysteine crosslinking approach to interrogate TM-TM relationships between (1) TCRβ and CD3γ subunits and (2) TCRα and ζ subunits. Taking these data together, I proposed a model of TCR-CD3 complex architecture. Elucidating the spatial arrangement of TCR-CD3 complex subunits has provided an instructive platform to understand how these components may cooperate to transmit the crucial signals that activate T cells