title: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, the Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem, 91120, Israel.
authors.forename: Prasanth
authors.initials: P
authors.lastname: Padala
authors.affiliation: Department of Biochemistry and Molecular Biology, the Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem, 91120, Israel.
authors.forename: Walaa
authors.initials: W
authors.lastname: Oweis
authors.affiliation: Department of Biochemistry and Molecular Biology, the Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem, 91120, Israel.
authors.forename: Bayan
authors.initials: B
authors.lastname: Mashahreh
authors.affiliation: Department of Biochemistry and Molecular Biology, the Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem, 91120, Israel.
authors.forename: Nadine
authors.initials: N
authors.lastname: Soudah
authors.affiliation: Department of Biochemistry and Molecular Biology, the Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem, 91120, Israel.
authors.forename: Einav
authors.initials: E
authors.lastname: Cohen-Kfir
authors.affiliation: Department of Chemistry and Biochemistry, James Madison University, Harrisonburg, Virginia, VA 22807, USA.
authors.forename: Emily A
authors.initials: EA
authors.lastname: Todd
authors.affiliation: Department of Chemistry and Biochemistry, James Madison University, Harrisonburg, Virginia, VA 22807, USA.
authors.forename: Christopher E
authors.identifier: http://orcid.org/0000-0002-4285-4341
authors.initials: CE
authors.lastname: Berndsen
authors.affiliation: Department of Biochemistry and Molecular Biology, the Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem, 91120, Israel. reuvenw@ekmd.huji.ac.il.
authors.forename: Reuven
authors.initials: R
authors.lastname: Wiener
mesh_terms: D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D044764:Ubiquitin-Activating Enzymes / Q000737:chemistry / Q000378:metabolism*
chemical_list: D011506:Proteins; C502095:UBA5 protein, human; C502094:UFM1 protein, human; D044764:Ubiquitin-Activating Enzymes