title: Mechanism-based small molecule cross-linkers of HECT E3 ubiquitin ligase-substrate pairs.
abstract: Here we report the discovery that bifunctional thiol- and amine-reactive electrophiles serve as mechanism-based covalent cross-linkers for HECT E3 ubiquitin ligase-substrate pairs. We demonstrate that these chemical cross-linkers covalently cross-link the catalytic Cys residue of the yeast HECT E3 ubiquitin ligase Rsp5 with the Lys of the ubiquitination site in the model substrate Sic60-GFP. This work represents the first example of a mechanism-based covalent cross-link of HECT E3-substrate pairs that converts transiently interacting HECT E3-substrate pairs into stable, covalently cross-linked protein complexes, thereby facilitating their subsequent isolation, identification, and study.
authors.affiliation: Department of Chemistry, Center for Molecular Innovation and Drug Discovery, Proteomics Center of Excellence, Chemistry of Life Processes Institute, Northwestern University, Silverman Hall, 2145 Sheridan Road, Evanston, IL 60208, USA.
authors.forename: Sungjin
authors.initials: S
authors.lastname: Park
authors.forename: Ioanna
authors.initials: I
authors.lastname: Ntai
authors.forename: Paul
authors.initials: P
authors.lastname: Thomas
authors.forename: Evgeniia
authors.initials: E
authors.lastname: Konishcheva
authors.forename: Neil L
authors.initials: NL
authors.lastname: Kelleher
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Statsuk
mesh_terms: D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000378:metabolism; D050756:Cyclin-Dependent Kinase Inhibitor Proteins / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D056827:Endosomal Sorting Complexes Required for Transport / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry; D010446:Peptide Fragments / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043743:Ubiquitin-Protein Ligase Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D003432:Cross-Linking Reagents; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D010446:Peptide Fragments; C086926:SIC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; C469287:RSP5 protein, S cerevisiae; D008239:Lysine; D003545:Cysteine