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.
Highlights
Modification of proteins by the addition of ubiquitin or ubiquitin-like proteins (UBLs) is crucial for proper functioning of cells
While Xie previously demonstrated that UBA5 that ends at amino acid 363 can activate UFM124, longer truncations have not been tested
While the above experiments have been done in the context of the adenylation domain, we were interested in whether UBA5 346 satisfies binding to Ubiquitin fold modifier 1 (UFM1) even in the absence of the adenylation domain
Summary
Modification of proteins by the addition of ubiquitin or ubiquitin-like proteins (UBLs) is crucial for proper functioning of cells. The addition of UBLs to target proteins, similar to ubiquitin, is carried out by a series of reactions. These reactions are catalyzed by three groups of enzymes, namely E1 (an activating enzyme), E2 (a conjugating enzyme) and E3 (a ligase enzyme). The activating enzyme E1 catalyzes the adenylation of the UBL C-terminus leading to thioester bond formation with the E1 active site cysteine. Eight E1 enzymes are known, each with specificity toward a particular UBL or ubiquitin These enzymes are classified into two groups: canonical and non-canonical activating enzymes[8]. In the activation process UBA5 adenylates UFM1 C-terminal Gly and forms a thioester bond with the UFM1 C-terminus via its active site Cys[250]. Together with the E3, UFL1, UFM1 is transferred to its target protein[20,21,22]
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