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.

Figure 1

UBA5 possesses a UFM1-binding sequence that is essential for UFM1 activation. (A) UBA5 constructs used in this study; AD is adenylation domain. (B) Charging assay of UBA5 constructs (10 μM) with UFM1 (50 μM). All constructs start at amino acid 57 and end as indicated by the number in the figure. (C) Pull down assay showing binding of UBA5 constructs (numbered as in B) to immobilized H₆-UFM1. (D) Pull down assay showing binding of GST-UBA5 constructs to immobilized H₆-UFM1. Experiment was performed as in C. (E) ConSeq evolutionary conservation analysis of UBA5. UBA5 residues located C-terminal to the adenylation domain are colored based on their conservation score. (F) Fluorescence polarization experiment showing binding of UFM1 to fluorescein-labeled UIS peptide K_D = 8.5 μM ± 2.3. Error bars represent the SEM of each measurement.

Figure 2

Structure of UIS-UFM1 complex. (A) Crystal structure of the UFM1 interacting sequence (UIS) of UBA5 fused to UFM1 (orange). The asymmetric unit contains two molecules A & B. Each molecule possesses a UFM1 part (orange) and a UIS part (blue). (B) Two asymmetric units possessing four molecules of UIS-UFM1 form a round structure where each molecule interacts with one molecule via the UFM1 part and another molecule via the UIS part. The two molecules that are colored as in A arrive from one asymmetric unit and the other two molecules come from adjacent asymmetric unit. (C) Superposition of the two UIS-UBA5 molecules in the asymmetric unit. Superposition was performed with the UFM1 part of each molecule. Colors are as in A.

Figure 3

Structural insight into the UIS-UFM1 interaction. (A) Contacts between UFM1 (orange) and the UIS of UBA5 (blue). (B) Electrostatic surface representation of UFM1 bound to the UIS. (C) Pull down assay showing the effect of UIS mutations on binding to UFM1. (D) Charging assay of UBA5 WT or mutants (20 μM) with UFM1 WT or mutants (100 μM).

Figure 4

UBA5 His 336 is involved in UFM1 binding and activation. (A,B) Steady-state kinetic curves of UBA5 (WT or mutant) charging with UFM1. Each rate was measured in triplicates and error bars indicate the SEM. (C,D) UBA5 Trp fluorescence intensity experiment showing binding of UBA5 WT or mutant to UFM1. Error bars represent the SEM of each measurement.

Figure 5

Structural analysis of UFM1 crystal structure. (A) Crystal structure of UFM1 showing the beta grasp fold commonly observed in ubiquitin and other UBLs. The asymmetric unit contains two molecules of UFM1. (B) Contact between two UFM1 molecules in asymmetric unit mimics the UIS binding to UFM1. Superposition between UFM1-UIS structure and UFM1 alone; the UIS is in blue. The spatial position of UIS H336 and I343 overlap with UFM1 H70 and I55, respectively. (C) Gel filtration elution profiles of UFM1 WT or mutants. (D) Superposition of UFM1 dimer (orange) with UBA5-UFM1 complex. UBA5 (red) holds the UFM1 dimer in an orientation that generates clashes with the UFM1 molecule that does not interact with the UIS and the adenylation domain, thereby preventing charging of UFM1 dimer.