Determining T-cell receptor binding orientation and Peptide-HLA interactions using cross-linking mass spectrometry

Area of Science:

• Immunology
• Structural Biology
• Biochemistry

Background:

• T cell receptors (TCRs) are vital for adaptive immunity, recognizing peptide-human leukocyte antigen (pHLA) complexes.
• Proper TCR-pHLA binding orientation is critical for T cell activation; incorrect orientations lead to non-functional T cells.
• Traditional methods for determining TCR-pHLA binding geometry, such as crystal structure determination, can be time-consuming.

Purpose of the Study:

• To introduce and validate a cross-linking tandem mass spectrometry (XL-MS/MS) method for elucidating TCR-pHLA binding orientation.
• To provide a faster and simpler alternative to existing structural determination techniques for TCR-pHLA interactions.

Main Methods:

• Application of a cross-linking tandem mass spectrometry (XL-MS/MS) approach to study TCR-pHLA complexes.
• Localization of cross-linking sites to specific amino acid residues within the TCR-pHLA interface.
• Validation of XL-MS/MS findings through structural prediction and crystal structure determination of selected TCR-pHLA complexes.

Main Results:

• The XL-MS/MS method successfully differentiated between canonical and reverse TCR binding orientations to pHLAs.
• Cross-linking sites provided detailed information on the molecular interactions at the TCR-pHLA interface.
• Structural predictions and experimental crystal structures confirmed the binding orientations determined by XL-MS/MS.

Conclusions:

• The developed XL-MS/MS method offers an efficient and accessible means to determine TCR-pHLA binding orientation.
• This technique facilitates a deeper understanding of T cell receptor interactions critical for immune responses and therapeutic development.