The T cell receptor (TCR)-CD3 complex is composed of a diverse TCR?? heterodimer noncovalently associated with the invariant CD3 dimers CD3??, CD3??, and CD3??. The TCR mediates peptide-MHC (pMHC) recognition, while the associated CD3 molecules transduce activation signals to the T cell. Whereas much is known about downstream T cell signaling, the mechanisms whereby TCR engagement by pMHC initiates signaling remain a mystery. Three basic mechanisms have been proposed: aggregation, segregation, and/or conformational change. A major attraction of the conformational change mechanism is that it can, in principle, account for triggering at very low densities of pMHC agonists on antigen-presenting cells. However, X-ray crystallographic studies have so far failed to identify clear and consistent conformational changes in the TCR C?? or C??domains that could be unambiguously attributed to antigen binding. One possibility is that such changes simply do not occur. Another is that crystal packing effects may have masked conformational changes in C?? or C? Yet a third possibility is that the relevant changes may be in protein dynamics, a parameter which cannot be accessed by X-ray crystallography. To distinguish among these possibilities, we propose to carry out the first solution NMR analysis of a full- length TCR ectodomain (V and C regions) in the unbound state and bound to pMHC. As our test system, we will use a human autoimmune TCR (MS2-3C8) that recognizes a peptide from myelin basic protein (MBP) and the MHC class II molecule HLA-DR4. The crystal structure of the MS2-3C8-MBP-DR4 complex, which we recently determined, revealed that TCR MS2-3C8 engages pMHC in the canonical docking mode of anti-foreign TCRs. Moreover, MS2-3C8 binds MBP-DR4 as tightly as the most avid anti- foreign TCRs. Our objectives are: 1. Structural analysis of free and pMHC-bound states of the TCR?? ectodomain in solution. We will determine the NMR solution structure of TCR MS2-3C8 in free form and bound to MBP-DR4. Because TCR is a heterodimer of ?- and ?-chains, separate isotope labeling of each chain will reduce spectral complexity and facilitate NMR analysis. Preliminary NMR spectra support the feasibility of achieving backbone RMSDs of <2.0 A and <3.0 A for individual domains and overall folds, respectively, which will permit a rigorous assessment of any ligand-induced conformational changes in MS2-3C8. 2. Backbone dynamics analysis of free and pMHC-bound states of the TCR?? ectodomain. Our goal here is to investigate whether pMHC-binding induces allosteric changes in TCR backbone dynamics. A wide time scale range will be examined (picoseconds to seconds) to assure detection of any alterations in backbone dynamics. Collectively, these studies should resolve whether the TCR undergoes long-range changes in conformation and/or dynamics in solution that can serve as a mechanism for T cell triggering.

Public Health Relevance

The mechanism by which TCR ligation leads to T cell triggering has remained a fundamental mystery in immunology for over 20 years. Three basic mechanisms have been suggested to explain how TCR ligation is communicated to the CD3 signaling apparatus: aggregation, segregation, and/or conformational change. Here, we address the conformational change hypothesis by proposing to carry out the first ever solution NMR analysis of a full-length TCR ectodomain (V and C regions) in the unbound state and bound to its peptide-MHC ligand, permitting detailed analyses that were not previously possible using other methods.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21AI097946-02
Application #
8416301
Study Section
Cellular and Molecular Immunology - B Study Section (CMIB)
Program Officer
Leitner, Wolfgang W
Project Start
2012-02-01
Project End
2014-01-31
Budget Start
2013-02-01
Budget End
2014-01-31
Support Year
2
Fiscal Year
2013
Total Cost
$190,000
Indirect Cost
$65,000
Name
University of Maryland College Park
Department
Miscellaneous
Type
Other Domestic Higher Education
DUNS #
790934285
City
College Park
State
MD
Country
United States
Zip Code
20742