Novel insights into the interaction of UBA5 with UFM1 via a UFM1-interacting sequence

Abstract

The modification of proteins by ubiquitin-fold modifier 1 (UFM1) is implicated in many human diseases. Prior to conjugation, UFM1 undergoes activation by its cognate activating enzyme, UBA5. UBA5 is a non-canonical E1 activating enzyme that possesses an adenylation domain but lacks a distinct cysteine domain. Binding of UBA5 to UFM1 is mediated via an amino acid sequence, known as the UFM1-interacting sequence (UIS), located outside the adenylation domain that is required for UFM1 activation. However, the precise boundaries of the UIS are yet not clear and are still under debate. Here we revisit the interaction of UFM1 with UBA5 by determining the crystal structure of UFM1 fused to 13 amino acids of human UBA5. Using binding and activity assays, we found that His 336 of UBA5, previously not reported to be part of the UIS, occupies a negatively charged pocket on UFM1’s surface. This His is involved in UFM1 binding and if mutated perturbs activation of UFM1. Surprisingly, we also found that the interaction between two UFM1 molecules mimics how the UIS binds UFM1. Specifically, UFM1 His 70 resembles UBA5 His336 and enters a negatively charged pocked on the other UFM1 molecule. Our results refine our understanding of UFM1-UBA5 binding.

An N-Terminal Extension to UBA5 Adenylation Domain Boosts UFM1 Activation: Isoform-Specific Differences in Ubiquitin-like Protein Activation

“…Previously, we and others have shown that the adenylation domain by itself is unable to activate UFM1, but a fragment possessing both the adenylation domain and the UIS successfully activates UFM1 [2,30]. Interestingly, although UBA5 dimerization is critical for UFM1 activation, UBA5 is not a stable dimer in solution.…”

“…Ubiquitin fold modifier 1 (UFM1) is a ubiquitin-like protein (UBL) that shares the β-grasp fold of ubiquitin even though it has only 16% sequence identity with ubiquitin [1][2][3].…”

“…Besides the adenylation domain, both UBA5 and Atg7 possess a sequence outside the adenylation domain that is required for UBL's binding. In UBA5 this sequence comprises 13 amino acids, which are known as the UFM1-interacting sequence (UIS) and are located C-terminal to the adenylation domain [2,29].…”

Trans ‐binding of UFM1 to UBA5 stimulates UBA5 homodimerization and ATP binding

“…However, Atg8 does not bind simultaneously to those 2 domains but, rather, first to the C‐terminal helical domain and then to the adenylation domain where Atg8 adenylation and the thioester bond formation take place. In contrast to Atg7, human UBA5 does not have a helical domain, but rather an ~13 aa sequence‐ located C‐terminal to the adenylation domain that serves as a UFM1‐interacting sequence (UIS) (23–25). Human UBA5 has 2 isoforms, both of which have the adenylation domain and the UIS, but only 1 has an extension of 56 aa N‐terminal to the adenylation domain, which was previously shown not to be essential for UBA5 activity (26, 27).…”

“…However, Atg8 does not bind simultaneously to those 2 domains but, rather, first to the C-terminal helical domain and then to the adenylation domain where Atg8 adenylation and the thioester bond formation take place. In contrast to Atg7, ABBREVIATIONS: Atg, autophagy-related; HEPES, 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid; ITC, isothermal titration calorimetric; K d , dissociation constant; SEC-MALS, size-exclusion chromatography with multiangle light scattering; UBA5, ubiquitin like modifier-activating enzyme 5; UBL, ubiquitin-like protein; UFC1, ubiquitin-fold modifier conjugating enzyme 1; UFL1, Ufm1 specific ligase 1; UFM1, ubiquitin fold modifier 1; UIS, UFM1-interacting sequence; WT, wild type human UBA5 does not have a helical domain, but rather an ;13 aa sequence-located C-terminal to the adenylation domain that serves as a UFM1-interacting sequence (UIS) (23)(24)(25). Human UBA5 has 2 isoforms, both of which have the adenylation domain and the UIS, but only 1 has an extension of 56 aa N-terminal to the adenylation domain, which was previously shown not to be essential for UBA5 activity (26,27).…”