Overall, S. | bioRxiv Science

bioRxiv2025

Intermolecular β-sheet formation guides the interaction between ubiquitin-like modifier FAT10 and adapter protein NUB1L

Under inflammatory conditions, the ubiquitin-like modifier FAT10 targets proteins for rapid and irreversible degradation by the 26S proteasome. FAT10 is degraded along with its substrates and in this process, the loose folding of FAT10 and adapter protein NUB1L have long been suspected to play crucial roles. We report here the investigation of the N-domain of FAT10 and its interaction with NUB1L by magic-angle spinning (MAS) NMR spectroscopy. A stretch of residues that is intrinsically disordered when the N-domain of FAT10 is in its ubiquitin-like {beta}-grasp fold, becomes part of a regularly structured loop and an intermolecular {beta}-sheet upon binding to NUB1L. The rest of the N-domain is now disordered, with exception of a series of anchor residues and the N-terminus. We propose that, in preparation of degradation by the proteasome, NUB1L stabilizes N-FAT10 in an unfolded state, acting as a holdase. The ability of FAT10 to interact in folded as well as unfolded form is essential for its role in inflammation-linked proteostasis.

biochemistry↗

bioRxiv2020

TAPBPR Promotes Antigen Loading on MHC-I Molecules Using a Peptide Trap

Chaperones tapasin and TAP-binding protein related (TAPBPR) perform the important functions of stabilizing nascent MHC-I molecules (chaperoning) and selecting high affinity peptides in the MHC-I groove (editing). While X-ray and cryo-EM snapshots of MHC-I in complex with TAPBPR and tapasin, respectively, have provided important insights into the peptide-deficient MHC-I groove structure, the molecular mechanism through which these chaperones influence the selection of specific amino acid sequences remains incompletely characterized. Of particular importance is a 16 residue loop in TAPBPR (corresponding to 11 residues in tapasin), which has been proposed to actively compete with incoming peptides by forming direct contacts in the F-pocket of the empty MHC-I groove. Using a deep mutational scanning functional analysis of TAPBPR, we find that important residues for the chaperoning activity are located on the major interaction surfaces with nascent MHC-I molecules, excluding the loop. However, interactions with properly conformed molecules toward peptide editing are influenced by loop mutations, in an MHC-I allele- and peptide-dependent manner. Detailed biophysical characterization by ITC, FP and NMR reveals that the loop does not interact with the peptide-deficient MHC-I groove to compete with incoming peptides, but instead promotes peptide loading by acting as a kinetic trap. Our results suggest that the longer loop of TAPBPR lowers the affinity threshold for peptide selection, to promote loading within subcellular compartments of reduced peptide concentration and to prevent disassembly of high affinity peptide-MHC-I complexes that are transiently interrogated by TAPBPR during editing.

immunology↗