The structure and catalytic mechanism of the Ub-like protein Uba5 ======= **Abstract** Regulation of biological processes often involves multiple enzymes progressing through mechanistic pathways to maintain life and homeostasis. One of these vital mechanisms is ubiquitination, a type of post-translational modification involved in the ufmylation cascade. Uba5 is a ubiquitin-like protein that acts as an E1 enzyme to catalyze the first steps of this process with Ufm1, a ubiquitin-fold modifier 1; however, the specific mechanism is unknown. Structural analysis in the program YASARA, BLAST alignment data with other species, and energy minimizations through the YASARA server were used to determine the structure, important residues, and effects of mutation on the Uba5 protein. After compiling the data from these experiments and the literature, a catalytic mechanism for Ufm1 activation leading to ufmylation by Uba5 was proposed. It was found that conserved residues across species remain important in the stabilization of both Ufm1 and Uba5 proteins to ensure proper ATP binding and subsequent activation of Ufm1 by Uba5. **Introduction** The human ubiquitin-activating enzyme 5 (UBA5) is an essential protein in the ufmylation cascade, a type of post-translational modification which controls cellular processes and is essential to survival of the cell. Modifications help to ensure the steady state in cells by regulating protein folding and interactions, and cell targeting *(1)*. Ubiquitination, a specific type of post-translational modification involves the addition of ubiquitin or ubiquitin like (Ubl) proteins to amine sidechains, and requires a set of three enzymes to complete the attachment. The activating enzyme (E1) first activates the Ubl, and then undergoes a transthioesterification reaction to transfer the Ubl to the conjugating enzyme (E2). Finally, the ligase enzyme (E3) transfers the Ubl to the substrate from the E2 (Figure 1). The human genome only codes a single ufmylation cascade (Uba5 – Ufci – Ufli), beginning with the E1 enzyme Uba5, as it acts on Ufm1 through the active site, Cys250, and the ATP binding site *(3)*. The E1 enzyme is very efficient, following activation by ATP, however prior to this binding, it has a low ubiquitin affinity, suggesting that the conformational change that occurs as a result of ATP binding helps propel the mechanism forward *(4)*. Binding in the adenylation domain stabilizes the homodimer, which places it in a favorable orientation for ATP binding *(5)*. The residues present in human Uba5 have been shown to be conserved across several species, indicating a high importance of these residues in binding to stabilize the molecules in a favorable orientation for binding and ufmylation to occur *(6)*. The function of this mechanism is well established, however the catalytic process is still largely unknown. This study aims to propose a plausible catalytic mechanism for the activation of Ufm1 by Uba5, by utilizing a stabilizing enzyme cave and favorable weak interactions between these two Ubls. @[osf](3s9gk) Figure 1. Model of Uba5 acting as an E1 enzyme in the ubiquitination pathway with Ufm1 and ATP *(1)*. **Methods** Structures of the Uba5 molecule as well as Ufm1 and ATP were analyzed in YASARA by identifying and visualizing the ATP binding site, Cys250 active site, and metal binding site. Following structural analysis, nearby residues were highlighted that could be used for stabilization both within the enzyme cave and between molecules of Uba5, Ufm1, and ATP *(7)*. Stabilizing residues surrounding the Cys250 active site included Uba5 V82, A111, C95, Y68, F276, and N210. Stabilizing residues between Uba5 and Ufm1 include Uba5 H215, Q217, and E209, and Ufm1 R79, D80, and R81. Bonds to AMP through the ATP active site were also modeled. Proposed mutations to these stabilizing residues were modeled and mutated, and new plausible bonds were formed using YASARA to assess favorability of each mutation or bond with regards to distance and weak interactions. These mutations included a Uba5 A111H mutation at the active site, a Uba5 Q209D mutation involved in stabilizing Ufm1, and a Uba5 G83Y mutation in the ATP binding site. These structures were all sent through the YASARA energy minimization server to correct the chemistry of the molecule as it would naturally occur in the aqueous environment of the human body at pH 7. Thermodynamic values, Z-scores, and presence or absence of formed bonds were compared between the control structure and the mutated structure, as well as within the same structure before and after the energy minimization. BLAST sequencing of Uba5 was used to then align five other species (*Glycine max, Caenorhabditis elegans, Drosophila mela, Mus musculs, and Danio rerio*) based on conserved residues in their protein sequences. These sequences and residues were then used to help support or disprove the importance of certain residues in the mechanism of ubiquitination. **Results** @[osf](pq3fh) Figure 1. Energy Minimization of Uba5-Ufm1 complex and ATP bound. Energy was recorded at -364789.1 kj/mol, and Z-score was recorded at -0.54. @[osf](42stq) Figure 2. Uba5 with Y68E mutation (red) surrounding the Cys250 active site (cyan). Other non-mutated surrounding residues used for stabilization are highlighted (orange, yellow, blue, green, and pink). @[osf](7df2e) Figure 3. Energy minimization of Uba5-Ufm1 complex with Y68E mutation in the ATP binding site. Energy was recorded at -363176.6 kj/mol, and Z-score was recorded at -0.41. @[osf](uthm4) Figure 4. Uba5 with A111H mutation (yellow) surrounding the Cys250 active site (cyan). Other non-mutated surrounding residues used for stabilization are highlighted (blue, green, pink, red, orange). @[osf](t9fnx) Figure 5. Energy minimization of Uba5-Ufm1 complex with A111H mutation in the ATP binding site. Energy was recorded at -361354.4 kj/mol, and Z-score was recorded at -0.43. @[osf](tejda) Figure 6. Energy minimization of Uba5-Ufm1 complex with Uba5-Ufm1 hydrogen bonds formed between H215-D80, Q217-R79, E209-R81, and R188-V82. Energy was recorded at -362422.4 kj/mol, and Z-score was recorded at -0.35. @[osf](wcx7y) Figure 7. Post-analysis of Uba5-Ufm1 hydrogen bond energy minimization displaying conserved, favorable bonds. Uba5 residues are colored in red, Ufm1 residues are colored cyan, and water molecules following energy minimization are colored as yellow spheres. @[osf](h6u9r) Figure 8. Uba5 Q209D mutation in hydrogen bonding with Ufm1. Ufm1 tail binding residues colored in yellow, and Uba5 Glutamic Acid mutation colored in magenta. @[osf](wncgv) Figure 9. Energy minimization of Uba5-Ufm1 complex with Uba5 Q209D mutation in hydrogen bonds. Energy was recorded at -361333.5 kj/mol, and Z-score was recorded at -0.42. @[osf](2w5xc) Figure 10. Uba5-AMP complex with formed bonds. AMP is highlighted in yellow, and Uba5 ATP binding residues are highlighted in red. @[osf](rygaz) Figure 11. Energy Minimization of Uba5-AMP complex with formed bonds. Energy was recorded at -363623.8 kj/mol, and Z-score was recorded at -0.39. @[osf](62fmr) Figure 12. Uba5-Ufm1 complex with Uba5 G83Y mutation bound to AMP. AMP highlighted in yellow, and Uba5 ATP binding site 83 highlighted in red with mutation shown. @[osf](kn6hf) Figure 13. Energy minimization of Uba5-Ufm1 complex with Uba5 G83Y mutation bound to AMP. Energy was recorded at -362322.9 kj/mol, and Z-score was recorded at -0.40. @[osf](d6xef) Figure 14. BLAST alignment of pdb code 5L95 with 5 different species, with conserves residues and sequences highlighted. @[osf](t95sx) Table 1. Energy minimization values following specific mutations on Uba5 including active site, ATP binding site and bonds formed between Uba5 and Ufm1. All delta G values are listed in kj/mol, and all Z-scores are standardized. **Conclusion** The catalytic mechanism (Figure 15) was proposed based on a compilation of data gathered through structural analysis of the ATP binding site and Cys250 active site on Uba5, as well as and Ufm1 binding residues. On the Ufm1 molecule, R81 and V82 are capable of forming hydrophobic interactions, and therefore can be stabilized by Ser or Ile in an enzyme cave to hold the molecule in place as it attacks the ATP *(8)*. Once it has attacked ATP, the Uba5 Cys250 active site can attack the D80 residue of the Ufm1, while it is stabilized by several nearby residues (Key Background - Figure 2) including Y68 hydrogen bonds to a Cys, F276 hydrophobic interactions and base stacking with another F, and V82 hydrophobic interactions with Ile in the enzyme cave. Once the Uba5 and Ufm1 are bound, several residues can interact to hold these in place, as was assessed through energy minimization to reveal the most favorable bonds. The following Uba5-Ufm1 bonds can occur to hold the molecule in place while acid-base chemistry acts upon the active site while Uba5 initiates ubiquitination: Q217-R79, H215-D80, and E209-R81. These bonds were formed and sent through the energy minimization server, and were found to be retained following the analysis (Figure 7). A mutation to the active site of G83Y would result in less favorable thermodynamic values as well as a less favorable z-score, indicating the G83 is important in the binding of ATP which is the initial activator of Uba5 to begin ubiquitination (Figure 12) (Figure 13). See Project Notebook for further explanation of each specific result). A mutation of G83Y was proposed to determine whether the mechanism proposed would be plausible. It was hypothesized that swapping a Glycene side chain which displays hydrophobic interactions with AMP to a Tyrosine side chain which has the potential to hydrogen could create more favorable thermodynamic interactions since the bonds are stronger, however the steric hinderance that would be experienced by the other residues in the ATP binding site would inhibit activation of Uba5 and ubiquitination; Therefore, it would have a less favorable structure as well. Therefore, this supports the hypothesis that these residues are plausible when bound together to create favorable weak interactions for the Uba5 and Ufm1 to interact and undergo ubiquitination. Although energy minimization is theoretically helpful, further analysis should be done. Circular Dichroism is a useful technique to assess secondary structure and whether folding favored the structure of Y83 over G83, or if it was even physically possible. If the CD results determine that it is unlikely, then the proposed mechanism (Figure 15) would be supported because it does not contain this mutation. Additionally, NMR spectroscopy would also be a useful technique to assess structure. It would help visualize magnetic fields around the molecule to determine whether functional groups were retained following binding. If the results determine that they were not, again the proposed mechanism (Figure 15) would be supported since the new functional group bond would not be favorable. @[osf](4c9gz) Figure 15. Proposed catalytic mechanism of Uba5-Ufm1 ufmylation cascade, with Uba5 acting as E1 enzyme. **Acknowledgements** Jasmine Brickey Laura Keane Monica Chiodo **References** (1) Berndsen, C. E. (2019) Modeled UBA5 Activation Pathway. (2) Tatsumi, K., Yamamoto-Mukai, H., Shimizu, R., Waguri, S., Sou, Y. S., Sakamoto, A., Taya, C., Shitara, H., Hara, T., Chung, C. H., Tanaka, K., Yamamoto, M., and Komatsu, M. (2011) The Ufm1-activating enzyme Uba5 is indispensable for erythroid differentiation in mice. Nat. Commun. 2, 181–187. (3) Wei, Y., and Xu, X. (2016) UFMylation: A Unique & Fashionable Modification for Life. Genomics. Proteomics Bioinformatics 14, 140–146. (4) Pickart, C. M. (2001) Mechanisms Underlying Ubiquitination. Annu. Rev. Biochem. 70, 503–33. 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