Periodic Reporting for period 1 - TCELLTIGIT (Structural characterization of immune signaling protein TIGIT using x-ray crystallography and cryo-electron microscopy)
Reporting period: 2019-01-01 to 2020-12-31
Immunosignaling proteins are the target of anti-cancer and anti-viral immunotherapies. These proteins are known as single pass transmembrane receptors and consist of 3 parts: an extracellular domain (ECD), a transmembrane (TM) domain, and an intracellular domain (ICD). The TM domain is important because it allows signals to be transferred from the outside of the cell to the inside through the cell membrane. However, very little is known about the TM domains of these proteins because no structures have been solved that include this domain. The objective of this project was to solve a structure of the immunosignaling protein TIGIT (T cell immunoreceptor with Ig and ITIM domains) that contains the TM domain. The goal of this was to allow us to better understand how these immunosignaling proteins conduct signals through the cell membrane. Understanding the mechanism for how these protein conduct signals through the membrane bilayer is important because it could potentially result in the design of new anti-cancer and anti-viral immunotherapies targeting the TM domains of these proteins. However, the action was terminated early due to medical issues experienced by the fellow, so we note that not all of the objectives of the action could be accomplished given the circumstances.
Work carried out on the project includes the following:
1: Chemical synthesis and purification of mammalian TIGIT TM peptides.
2: Bacterial expression and purification of human TIGIT constructs.
3: Crystallization trials.
4: Diffraction trials.
5: Data analysis.
The TM domain of TIGIT is small enough to be chemically synthesized, so this approach was used to produce mammalian TIGIT TM peptide sample for crystallization trials. HPLC purification was used to ensure that the peptide sample was sufficiently pure. Bacterial expression of several human TIGIT constructs was investigated. For one TIGIT construct containing the ECD and TM domains, sample preparation and purification methods were optimized to the point of producing bacterially expressed protein sample that was pure enough for crystallization trials. Crystallization trials were carried out using the mammalian TIGIT TM peptide and human TIGIT ECDTM construct, primarily using lipid cubic phase (LCP) crystallization techniques. Diffraction trials were carried out at Diamond Light Source, and the data resulting from these trials was analyzed in an attempt to solve atomic resolution structures. These results were disseminated internally through meetings and discussions with colleagues at Trinity College Dublin, and were presented informally to the public at Trinity PROBE night.
1: Chemical synthesis and purification of mammalian TIGIT TM peptides.
2: Bacterial expression and purification of human TIGIT constructs.
3: Crystallization trials.
4: Diffraction trials.
5: Data analysis.
The TM domain of TIGIT is small enough to be chemically synthesized, so this approach was used to produce mammalian TIGIT TM peptide sample for crystallization trials. HPLC purification was used to ensure that the peptide sample was sufficiently pure. Bacterial expression of several human TIGIT constructs was investigated. For one TIGIT construct containing the ECD and TM domains, sample preparation and purification methods were optimized to the point of producing bacterially expressed protein sample that was pure enough for crystallization trials. Crystallization trials were carried out using the mammalian TIGIT TM peptide and human TIGIT ECDTM construct, primarily using lipid cubic phase (LCP) crystallization techniques. Diffraction trials were carried out at Diamond Light Source, and the data resulting from these trials was analyzed in an attempt to solve atomic resolution structures. These results were disseminated internally through meetings and discussions with colleagues at Trinity College Dublin, and were presented informally to the public at Trinity PROBE night.
Because the action was terminated before its scheduled end date, we were unable to make progress beyond the state of the art or expected results.