A Concerted Action of UBA5 C-Terminal Unstructured Regions Is Important for Transfer of Activated UFM1 to UFC1.

Nicole Wesch,Frank Löhr,Natalia Rogova,Volker Dötsch,Vladimir V Rogov

doi:10.3390/ijms22147390

Nicole Wesch, Frank Löhr + Show 3 more

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https://doi.org/10.3390/ijms22147390

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Abstract

Ubiquitin fold modifier 1 (UFM1) is a member of the ubiquitin-like protein family. UFM1 undergoes a cascade of enzymatic reactions including activation by UBA5 (E1), transfer to UFC1 (E2) and selective conjugation to a number of target proteins via UFL1 (E3) enzymes. Despite the importance of ufmylation in a variety of cellular processes and its role in the pathogenicity of many human diseases, the molecular mechanisms of the ufmylation cascade remains unclear. In this study we focused on the biophysical and biochemical characterization of the interaction between UBA5 and UFC1. We explored the hypothesis that the unstructured C-terminal region of UBA5 serves as a regulatory region, controlling cellular localization of the elements of the ufmylation cascade and effective interaction between them. We found that the last 20 residues in UBA5 are pivotal for binding to UFC1 and can accelerate the transfer of UFM1 to UFC1. We solved the structure of a complex of UFC1 and a peptide spanning the last 20 residues of UBA5 by NMR spectroscopy. This structure in combination with additional NMR titration and isothermal titration calorimetry experiments revealed the mechanism of interaction and confirmed the importance of the C-terminal unstructured region in UBA5 for the ufmylation cascade.

Highlights

Ubiquitin fold modifier 1 (UFM1) is a small ubiquitin-like (UBL) protein spanning 85 residues
The results indicate that the LIR/UFIM sequence is more important for the ufmylation cascade than the R3 site and that the conserved region R2 could play an additive role in this process: the level of UFC1~UFM1 conjugates reached in reactions with AD1–330/R1-R2325–376 a higher level than when the R1325–357 peptide was added alone
Ability of the isolated UBA5 AD to transfer activated UFM1 on UFC1 gets rescued by addition of the R1-R2-R3 peptide

Summary

Introduction

UFM1 is a small ubiquitin-like (UBL) protein spanning 85 residues. Like other UBLs, it has a low sequence identity to ubiquitin, but shares its specific (β-grasp) fold [1,2]. The UFM1 precursor protein gets processed by the two specific proteases UfSP1 and UfSP2 to expose the C-terminal glycine residue [5,6,7]. The last step is the transfer of UFM1 to the target proteins mediated by the specific UFM1 ligase 1 (UFL1), showing no typical E3 ligases domain organization [1,12]. The mechanism of this step is largely unknown and other proteins could be required for UFL1 ligase activity as well [13,14,15,16]

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