Az orvos-, egészségtudományi- és gyógyszerészképzés tudományos műhelyeinek fejlesztése(EFOP-3.6.3-VEKOP-16-2017-00009)
Támogató: EFOP-VEKOP
(NTP-NFTÖ-P-15-0693) Támogató: Emberi Erőforrások Minisztériuma
(NTP-NFTÖ-P-17-C-0139) Támogató: Emberi Erőforrások Minisztériuma
(NTP-NFTÖ-P-19-B-0270) Támogató: Emberi Erőforrások Minisztériuma
Szakterületek:
Biokémia és molekuláris biológia
T lymphocytes discriminate between healthy versus infected or cancerous cells via
T-cell receptor mediated recognition of peptides bound and presented by cell-surface-expressed
major histocompatibility complex molecules (MHCs). Pre-T cell receptors (preTCRs)
on thymocytes foster development of αβT lymphocytes through their β chain interaction
with MHC displaying self-peptides on thymic epithelia. The specific binding of a preTCR
with a peptide-MHC complex (pMHC) has been identified previously as forming a weak
affinity complex with a distinct interface from that of mature αβTCR. However, a lack
of appropriate tools has limited prior efforts to investigate this unique interface.
Here we designed a small-scale linkage screening protocol using bismaleimide linkers
for determining residue-specific distance constraints between transiently interacting
protein pairs in solution. Employing linkage distance restraint-guided molecular modeling,
we report the oriented solution docking geometry of a preTCRβ-pMHC interaction. The
linkage model of preTCRβ-pMHC complex was independently verified with paramagnetic
pseudocontact chemical shift (PCS) NMR of the unlinked protein mixtures. Using linkage
screens, we show that the preTCR binds with differing affinities to peptides presented
by MHC in solution. Moreover, the C-terminal peptide segment is a key determinant
in preTCR-pMHC recognition. We also describe the process for future large-scale production
and purification of the linked constructs for NMR, X-ray crystallography and single
molecule electron microscopy studies.