title: Mechanistic study of Uba5 enzyme and the Ufm1 conjugation pathway.
abstract: E1 enzymes activate ubiquitin or ubiquitin-like proteins (Ubl) via an adenylate intermediate and initiate the enzymatic cascade of Ubl conjugation to target proteins or lipids. Ubiquitin-fold modifier 1 (Ufm1) is activated by the E1 enzyme Uba5, and this pathway is proposed to play an important role in the endoplasmic reticulum (ER) stress response. However, the mechanisms of Ufm1 activation by Uba5 and subsequent transfer to the conjugating enzyme (E2), Ufc1, have not been studied in detail. In this work, we found that Uba5 activated Ufm1 via a two-step mechanism and formed a binary covalent complex of Uba5∼Ufm1 thioester. This feature contrasts with the three-step mechanism and ternary complex formation in ubiquitin-activating enzyme Uba1. Uba5 displayed random ordered binding with Ufm1 and ATP, and its ATP-pyrophosphate (PPi) exchange activity was inhibited by both AMP and PPi. Ufm1 activation and Uba5∼Ufm1 thioester formation were stimulated in the presence of Ufc1. Furthermore, binding of ATP to Uba5∼Ufm1 thioester was required for efficient transfer of Ufm1 from Uba5 to Ufc1 via transthiolation. Consistent with the two-step activation mechanism, the mechanism-based pan-E1 inhibitor, adenosine 5'-sulfamate (ADS), reacted with the Uba5∼Ufm1 thioester and formed a covalent, tight-binding Ufm1-ADS adduct in the active site of Uba5, which prevented further substrate binding or catalysis. ADS was also shown to inhibit the Uba5 conjugation pathway in the HCT116 cells through formation of the Ufm1-ADS adduct. This suggests that further development of more selective Uba5 inhibitors could be useful in interrogating the roles of the Uba5 pathway in cells.
authors: Oncology Drug Discovery Unit, Takeda Pharmaceuticals International Co., Cambridge, Massachusetts 02139. Electronic address: james.gavin@takeda.com.
authors: James M
authors: JM
authors: Gavin
authors: Oncology Drug Discovery Unit, Takeda Pharmaceuticals International Co., Cambridge, Massachusetts 02139.
authors: Kara
authors: K
authors: Hoar
authors: Oncology Drug Discovery Unit, Takeda Pharmaceuticals International Co., Cambridge, Massachusetts 02139.
authors: Qing
authors: Q
authors: Xu
authors: Oncology Drug Discovery Unit, Takeda Pharmaceuticals International Co., Cambridge, Massachusetts 02139.
authors: Jingya
authors: J
authors: Ma
authors: Oncology Drug Discovery Unit, Takeda Pharmaceuticals International Co., Cambridge, Massachusetts 02139.
authors: Yafang
authors: Y
authors: Lin
authors: Oncology Drug Discovery Unit, Takeda Pharmaceuticals International Co., Cambridge, Massachusetts 02139.
authors: Jiejin
authors: J
authors: Chen
authors: Oncology Drug Discovery Unit, Takeda Pharmaceuticals International Co., Cambridge, Massachusetts 02139.
authors: Wei
authors: W
authors: Chen
authors: Oncology Drug Discovery Unit, Takeda Pharmaceuticals International Co., Cambridge, Massachusetts 02139.
authors: Frank J
authors: FJ
authors: Bruzzese
authors: Oncology Drug Discovery Unit, Takeda Pharmaceuticals International Co., Cambridge, Massachusetts 02139.
authors: Sean
authors: S
authors: Harrison
authors: Oncology Drug Discovery Unit, Takeda Pharmaceuticals International Co., Cambridge, Massachusetts 02139.
authors: William D
authors: WD
authors: Mallender
authors: Oncology Drug Discovery Unit, Takeda Pharmaceuticals International Co., Cambridge, Massachusetts 02139.
authors: Nancy J
authors: NJ
authors: Bump
authors: Oncology Drug Discovery Unit, Takeda Pharmaceuticals International Co., Cambridge, Massachusetts 02139.
authors: Michael D
authors: MD
authors: Sintchak
authors: Oncology Drug Discovery Unit, Takeda Pharmaceuticals International Co., Cambridge, Massachusetts 02139.
authors: Neil F
authors: NF
authors: Bence
authors: Oncology Drug Discovery Unit, Takeda Pharmaceuticals International Co., Cambridge, Massachusetts 02139.
authors: Ping
authors: P
authors: Li
authors: Oncology Drug Discovery Unit, Takeda Pharmaceuticals International Co., Cambridge, Massachusetts 02139.
authors: Lawrence R
authors: LR
authors: Dick
authors: Oncology Drug Discovery Unit, Takeda Pharmaceuticals International Co., Cambridge, Massachusetts 02139.
authors: Alexandra E
authors: AE
authors: Gould
authors: Oncology Drug Discovery Unit, Takeda Pharmaceuticals International Co., Cambridge, Massachusetts 02139. Electronic address: jesse.chen@takeda.com.
authors: Jesse J
authors: JJ
authors: Chen
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D008956:Models, Chemical; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044764:Ubiquitin-Activating Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D046912:Multiprotein Complexes; D011506:Proteins; C502095:UBA5 protein, human; C502096:UFC1 protein, human; C502094:UFM1 protein, human; D000255:Adenosine Triphosphate; D044763:Ubiquitin-Conjugating Enzymes; D044764:Ubiquitin-Activating Enzymes
keywords: Endoplasmic Reticulum (ER); Endoplasmic Reticulum Stress (ER Stress); Enzyme Inactivation; Enzyme Inhibitor; Enzyme Inhibitors; Enzyme Mechanisms; Substrate-assisted Inhibitors; Uba5; Ubiquitin-conjugating Enzyme (E2 enzyme); Ufc1; Ufm1