Co-sedimentation is the key to the structural investigation of wild-type FAT10
Under inflammatory conditions, the ubiquitin-like modifier FAT10 serves as a tag for protein degradation by the 26S proteasome. FAT10 is degraded along with its substrates and this process is independent of the segregase VCP/p97, which, in the regular ubiquitin pathway of degradation, is required if a substrate lacks a disordered initiation region. FAT10 itself is loosely folded and its tendency to aggregate has complicated investigations of its structure, interaction, and function. Recently hydrogen-deuterium exchange in combination with mass spectrometry has suggested that, in preparation of degradation by the proteasome, the adapter protein NUB1 traps FAT10 in a mostly unfolded state by capturing a {beta}-strand. {beta}-strand capture was subsequently confirmed by magic-angle spinning (MAS) NMR spectroscopy of a stabilized variant of the N-domain of FAT10 in complex with NUB1L, the longer splice variant of NUB1. MAS NMR, in addition, revealed that the N-domain of FAT10 and NUB1L form a fuzzy complex and that the N-terminus of FAT10 is positioned for initiation of degradation by specific non-covalent interaction with NUB1L. Here, we report the investigation of the wild-type N-domain of FAT10 by MAS NMR. Co-sedimentation with NUB1L yields high-quality spectra, which enable sequential assignment of resonances. Through the lens of MAS NMR, the complexes of the wild-type and stabilized N-domain of FAT10 with NUB1L are identical. The N-terminus of FAT10 again shows up prominently in the spectra, even though the residue is this time an Ala, not a Gly. Our experience suggests that co-sedimentation in combination with MAS NMR is generally helpful in the exploration of conditional folds of intrinsically disordered proteins.