Abstract
FAT10 is a ubiquitin-like modifier that directly targets proteins for proteasomal degradation. Here, we report the high-resolution structures of the two individual ubiquitin-like domains (UBD) of FAT10 that are joined by a flexible linker. While the UBDs of FAT10 show the typical ubiquitin-fold, their surfaces are entirely different from each other and from ubiquitin explaining their unique binding specificities. Deletion of the linker abrogates FAT10-conjugation while its mutation blocks auto-FAT10ylation of the FAT10-conjugating enzyme USE1 but not bulk conjugate formation. FAT10- but not ubiquitin-mediated degradation is independent of the segregase VCP/p97 in the presence but not the absence of FAT10’s unstructured N-terminal heptapeptide. Stabilization of the FAT10 UBDs strongly decelerates degradation suggesting that the intrinsic instability of FAT10 together with its disordered N-terminus enables the rapid, joint degradation of FAT10 and its substrates without the need for FAT10 de-conjugation and partial substrate unfolding.
Highlights
FAT10 is the only UBL modifier to directly target proteins for proteasomal degradation independently of Ub attachment[12,13,14,15,16]
To improve the long-term stability of the wild-type (WT) human FAT10 protein, we replaced the four cysteines in FAT10 with Ser, Thr, or Leu depending on whether they were predicted by bioinformatics analysis to be solvent-exposed or buried (C7T and C9T in the Ndomain; C134L and C162S in the C-domain) (Fig. 1a)
We detected only few amino acids in the FAT10 C0 protein that gave rise to small magnitude (
Summary
FAT10 is the only UBL modifier to directly target proteins for proteasomal degradation independently of Ub attachment[12,13,14,15,16]. The FAT10 N- and C-domains share 29% and 36% identity with Ub for the human proteins, respectively, and only 18% identity with each other Both mouse and human FAT10 are poorly soluble in mammalian cells and when overexpressed in E. coli[21,22]. We have succeeded in increasing the solubility of full-length human FAT10 and the separate N- and C-domains This allowed us to determine the high-resolution structures of the individual FAT10 domains by x-ray crystallography and solution-state NMR spectroscopy. Based on this structural information, we performed experiments on the role of the domain linker, the individual UBDs and the disordered N-terminal extension for FAT10 conjugation and for FAT10-mediated proteasomal degradation. Our data suggest that the unstructured N-terminal heptapeptide together with the poor stability of FAT10 enables a fast, direct, and irreversible targeting of FAT10 along with its substrates to the 26S proteasome without the need for partial substrate unfolding by VCP and cleavage of FAT10 prior to degradation
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