# Understanding the structural basis of T cell receptor (TCR) and preTCR mechanosensing: single molecule, NMR and molecular dynamics studies

> **NIH NIH R01** · VANDERBILT UNIVERSITY · 2022 · $743,539

## Abstract

ABSTRACT
The mammalian adaptive immune system protects its host against infectious diseases as well as tumors in a
highly specific manner. At the core of ab T lymphocyte recognition is self- vs. non-self-discrimination, a
functionality endowed by clonal cell-surface T cell receptors (TCRs). The millions of distinct TCRs expressed in
the mammalian thymus create a repertoire that is refined to eliminate unwanted autoreactive specificities prior
to export into the peripheral lymphoid compartment. Once there, mature abT cells scan their environment
during immune surveillance, generating tensile and shear stresses over a wide range of pN-nN forces. Direct
evidence that the TCR acts as a mechanosensor has been provided, explaining its exquisite specificity and
sensitivity yet low affinity for ligand in the absence of physical load. Recently, we showed that force-based
abTCR discrimination extended to its developmental precursor, the preTCR, a pTa-b heterodimer. Moreover,
reversible structural rearrangements necessary for strengthened ligand binding under force were observed in
both TCR and preTCR. In this proposal, we shall combine single molecule (SM) and single cell (SMSC)
methods using optical traps, structure-function mutational analyses, recombinant protein expression and
molecular dynamic simulation to probe TCR and preTCR complexes with pMHC under load to provide a clear
understanding for the structural basis of mechanosensing. It is our hypothesis that binding is "gated" for
unloaded TCRs but that TCRs enter a "binding reading state" when force loaded, extend and either stabilize
the bond with lifetime lengthening to facilitate signaling or, alternatively, quickly release from irrelevant ligands.
In Aim 1, we will elucidate the critical TCR a and b subunit variable (V) and constant (C) domain structural
elements including the Cb FG loop involved in mechanically modulating the strength of loaded TCR-pMHC
interactions. Topologically stabilized structures as well as de-stabilizing mutations will be assessed for their
ability to alter pMHC bond lifetime and conformational change as well as to impact ab T cell activation as
measured by cytokine production. We will leverage newly developed single molecule and single tether assays
for direct comparison of strength of loaded TCRs on isolated TCRab-pMHC complexes and ab T cell lines. Aim
2 will examine mechanical tuning of the preTCR and how preTCR pTa-b structures differ from those of TCRab.
In both Aims 1 and 2, we will identify conformational transitions leading to bond strengthening and release
pathways critical to T cell activation and development. The effects of preTCR mutations on thymocyte
developmental progression will be determined experimentally using the thymic stromal cell line OP9-DL4 and
fetal liver hematopoietic progenitors transduced with wild-type or mutant preTCRs. Aim 3 will employ in silico
molecular dynamics simulation to identify unfolding pathways of TCR-pMHC or preTCR-pMHC compl...

## Key facts

- **NIH application ID:** 10406149
- **Project number:** 5R01AI136301-05
- **Recipient organization:** VANDERBILT UNIVERSITY
- **Principal Investigator:** Matthew J. Lang
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $743,539
- **Award type:** 5
- **Project period:** 2018-06-22 → 2025-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10406149

## Citation

> US National Institutes of Health, RePORTER application 10406149, Understanding the structural basis of T cell receptor (TCR) and preTCR mechanosensing: single molecule, NMR and molecular dynamics studies (5R01AI136301-05). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10406149. Licensed CC0.

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