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Updated: Jun 10, 2026
Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation
Published on: February 28, 2019
Combinations of affinity-enhancing mutations in a T cell receptor reveal highly nonadditive effects within and
Brian G Pierce1, Jaafar N Haidar, Yong Yu
1Bioinformatics Program, Boston University, Boston, Massachusetts 02215, USA.
Abstract:
Understanding the energetic and structural response to multiple mutations in a protein-protein interface is a key aspect of rational protein design. Here we investigate the cooperativity of combinations of point mutations of a T cell receptor (TCR) that binds in vivo to HLA-A2 MHC and a viral peptide. The mutations were obtained from two sources: a structure-based design study on the TCR alpha chain (nine mutations) and an in vitro selection study on the TCR beta chain (four mutations). In addition to combining the highest-affinity variants from each chain, we tested other combinations of mutations within and among the chains, for a total of 23 TCR mutants that we measured for binding kinetics to the peptide and major histocompatibility complex. A wide range of binding affinities was observed, from 2- to 1000-fold binding improvement versus that of the wild type, with significant nonadditive effects observed within and between TCR chains. This included an amino acid-dependent cooperative interaction between CDR1 and CDR3 residues that are separated by more than 9 A in the wild-type complex. When analyzing the kinetics of the mutations, we found that the association rates were primarily responsible for the cooperativity, while the dissociation rates were responsible for the anticooperativity (less-than-additive energetics). On the basis of structural modeling of anticooperative mutants, we determined that side chain clash between proximal mutants likely led to nonadditive binding energies. These results highlight the complex nature of TCR association and binding and will be informative in future design efforts that combine multiple mutant residues.
Insights
Investigating T cell receptor (TCR) mutations reveals complex binding interactions. Multiple mutations show significant nonadditive effects, impacting protein design strategies for TCRs and MHC binding.
Area of Science:
- Immunology
- Structural Biology
- Protein Engineering
Background:
- Understanding protein-protein interactions is crucial for rational protein design.
- T cell receptors (TCRs) play a vital role in adaptive immunity by recognizing peptide-MHC complexes.
- Investigating mutation effects on TCR binding affinity is key for therapeutic development.
Purpose of the Study:
- To investigate the energetic and structural consequences of combining multiple mutations in a T cell receptor (TCR).
- To understand the cooperative and nonadditive effects of mutations on TCR binding affinity to HLA-A2 MHC and viral peptides.
- To inform future protein design strategies by elucidating the complexities of TCR-peptide-MHC interactions.
Main Methods:
- Generated 23 T cell receptor (TCR) mutants by combining point mutations from structure-based and in vitro selection studies.
- Measured binding kinetics (affinity, association, and dissociation rates) of TCR mutants to HLA-A2 MHC and viral peptide.
- Utilized structural modeling to analyze anticooperative mutants and identify the structural basis for nonadditive binding energies.
Main Results:
- Observed a wide range of binding affinities, with improvements of 2- to 1000-fold compared to wild-type TCR.
- Identified significant nonadditive (cooperative and anticooperative) effects within and between TCR chains.
- Found that altered association rates drove cooperativity, while altered dissociation rates contributed to anticooperativity, likely due to side chain clashes.
Conclusions:
- TCR binding affinity is influenced by complex, nonadditive interactions between multiple mutations.
- Amino acid-dependent cooperative interactions can occur between residues distant in the wild-type complex.
- Understanding these intricate binding dynamics is essential for successful protein engineering and therapeutic design involving TCRs.
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