Suggestions for changes to GO’s “ubiquitin” MF terms

1. Suggestions for changes to GO’s “ubiquitin” MF terms Mike Livstone Working with Rose Oughtred and Chandra Theesfeld On behalf of BioGRID and UbiGRID

2. Background: Protein ubiquitylation Activation: E1 + Ub + ATP  E1-Ub + AMP + PPi (thioester bond) Conjugation (transthiolation): E1-Ub + E2  E2-Ub + E1 (thioester bond) Ligation: E2-Ub + E3 + S  S-Ub + E2 + E3 (isopeptide bond) Occurs w/w/o transfer to E3 Metzger et al., J. CellSci. 125:531-7 (2012)

3. Issue # 1: Only 2 relevant MF terms currently in GO Remaining terms describe: • Binding • Removal of Ub • Regulation • Child terms E2, E3 E1

4. Issue #2:The definitions are essentially identical GO:0004839 ubiquitin activating enzyme activity Definition: Catalysis of the reaction: a protein L-lysine + a ubiquitin + ATP = a protein-N-ubiquityl-lysine + AMP + diphosphate. This reaction is the activation of the C-terminal carboxyl group of ubiquitin by the formation of a high-energy thiolester bond in an ATP-dependent manner. GO:0004842 ubiquitin-protein ligase activity Definition: Catalysis of the reaction: ATP + ubiquitin + protein lysine = AMP + diphosphate+ protein N-ubiquityllysine.

5. Issue #3:ATP hydrolysis E1 + Ub + ATP E1-Ub + AMP + PPi GO:0004839, “ubiquitin activating enzyme activity” Definition: Catalysis of the reaction: a protein L-lysine + a ubiquitin + ATP = a protein-N-ubiquityl-lysine + AMP + diphosphate. This reaction is the activation of the C-terminal carboxyl group of ubiquitin by the formation of a high-energy thiolester bond in an ATP-dependent manner. GO:0004842, “ubiquitin-protein ligase activity” Definition: Catalysis of the reaction: ATP + ubiquitin + protein lysine = AMP + diphosphate+ protein N-ubiquityllysine. E1-Ub + E2  E2-Ub + E1 E2-Ub + E3 + S  S-Ub + E2 + E3 GO:0016874, “ligase activity” Catalysis of the joining of two substances, or two groups within a single molecule, with the concomitant hydrolysis of the diphosphate bond in ATP or a similar triphosphate.

6. Suggestions(specifics on following slides) • Fix the definition and parentage of GO:4842 to remove the requirement for ATP. • Split GO:4842 into two terms to differentiate between the different types of bonds. • Update the definition of GO:4839 to be consistent with the above.

7. Part 1: Revised definition forGO:0004842, “ubiquitin-protein ligase activity”(E2-Ub + E3 + S  S-Ub + E2 + E3) Current definition: Catalysis of the reaction: ATP + ubiquitin + protein lysine = AMP + diphosphate + protein N-ubiquityllysine. Proposed definition: Attachment of ubiquitin to a protein substrate via the reaction X-Ub + S  X + Ub-S, where X is either an E2 or E3 enzyme, the X-Ub linkage is a thioester bond, and the S-Ub linkage is an isopeptide bond between the C-terminal glycine of ubiquitin and the epsilon-amino group of lysine residues in the substrate. Note that this may include the extension of ubiquitin chains via ubiquitination of previously ubiquitinated proteins. part_of GO:0016567 protein ubiquitination [currently in place] Remove is_a “small conjugating protein ligase activity” (requires ATP) ??? ADD is_a GO:0016740, “transferase activity” Covers transfer from E2 or E3 w/w/o any accessory proteins.

8. Part 2: A new term to describeE1-Ub + E2  E2-Ub + E1 GO:NEW, “Ubiquitin transthiolation activity” Synonym: Ubiquitin conjugating enzyme activity Definition: (“Isoenergetic” - optional) transfer of ubiquitin from one protein to another via the reaction X-ubiquitin + Y → Y-ubiquitin + X, where both the X-ubiquitin and Y-ubiquitin linkages are thioester bonds between the C-terminal glycine of ubiquitin and a sulfhydryl side group of a cysteine residue. ??? is_a GO:0016740, “transferase activity” [“Catalysis of the transfer of a group, e.g. a methyl group, glycosyl group, acyl group, phosphorus-containing, or other groups, from one compound (generally regarded as the donor) to another compound (generally regarded as the acceptor). Transferase is the systematic name for any enzyme of EC class 2.”] part_of GO:0016567, “protein ubiquitination” Also covers E2-Ub + E3  E3-Ub + E2

9. Part 3: Revised definition forGO:0004839, “ubiquitin activating enzyme activity”(E1 + Ub + ATP  E1-Ub + AMP + PPi) Current definition: Catalysis of the reaction: a protein L-lysine + a ubiquitin + ATP = a protein-N-ubiquityl-lysine + AMP + diphosphate. This reaction is the activation of the C-terminal carboxyl group of ubiquitin by the formation of a high-energy thiolester bond in an ATP-dependent manner. Proposed definition: Catalysis of the reaction: E1 + ubiquitin + ATP  E1-ubiquitin + AMP + PPi, where the E1-ubiquitin linkage is a thioester bond between the C-terminal glycine of Ub and a sulfhydryl side group of an E1 cysteine residue. This is the first step in a cascade of reactions in which ubiquitin is ultimately added to a protein substrate. Synonym: E1 [currently in place] is_a GO:0008641, “small protein activating enzyme activity” [currently in place] ADD is_a GO:0016877, “ligase activity, forming carbon-sulfur bonds” ADD part_of GO:0016567, “protein ubiquitination”

10. Outlook • MF: 3 suggestions as described • Potentially parallel suggestions for sumoylation and/or neddylation • CC: no suggestions • BP: No issues, but considering some ways to flesh out some of the E2/E3 paths How should we proceed?