title: Two ZnF-UBP domains in isopeptidase T (USP5).
abstract: Human ubiquitin-specific cysteine protease 5 (USP5, also known as ISOT and isopeptidase T), an 835-residue multidomain enzyme, recycles ubiquitin by hydrolyzing isopeptide bonds in a variety of unanchored polyubiquitin substrates. Activation of the enzyme's hydrolytic activity toward ubiquitin-AMC (7-amino-4-methylcoumarin), a fluorogenic substrate, by the addition of free, unanchored monoubiquitin suggested an allosteric mechanism of activation by the ZnF-UBP domain (residues 163-291), which binds the substrate's unanchored diglycine carboxyl tail. By determining the structure of full-length USP5, we discovered the existence of a cryptic ZnF-UBP domain (residues 1-156), which was tightly bound to the catalytic core and was indispensable for catalytic activity. In contrast, the previously characterized ZnF-UBP domain did not contribute directly to the active site; a paucity of interactions suggested flexibility between these two domains consistent with an ability by the enzyme to hydrolyze a variety of different polyubiquitin chain linkages. Deletion of the known ZnF-UBP domain did not significantly affect rate of hydrolysis of ubiquitin-AMC and suggested that it is likely associated mainly with substrate targeting and specificity. Together, our findings show that USP5 uses multiple ZnF-UBP domains for substrate targeting and core catalytic function.
authors.affiliation: Structural Genomics Consortium, University of Toronto, Toronto, ON M5G 1L7, Canada.
authors.forename: George V
authors.initials: GV
authors.lastname: Avvakumov
authors.forename: John R
authors.initials: JR
authors.lastname: Walker
authors.forename: Sheng
authors.initials: S
authors.lastname: Xue
authors.forename: Abdellah
authors.initials: A
authors.lastname: Allali-Hassani
authors.forename: Abdalin
authors.initials: A
authors.lastname: Asinas
authors.forename: Usha B
authors.initials: UB
authors.lastname: Nair
authors.forename: Xianyang
authors.initials: X
authors.lastname: Fang
authors.forename: Xiaobing
authors.initials: X
authors.lastname: Zuo
authors.forename: Yun-Xing
authors.initials: YX
authors.lastname: Wang
authors.forename: Keith D
authors.initials: KD
authors.lastname: Wilkinson
authors.forename: Sirano
authors.initials: S
authors.lastname: Dhe-Paganon
mesh_terms: D019759:Carbon-Nitrogen Lyases / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D005459:Fluorides / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D013379:Substrate Specificity; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism; D054875:Ubiquitination; D017967:Zinc Compounds / Q000737:chemistry* / Q000378:metabolism
chemical_list: D025801:Ubiquitin; D017967:Zinc Compounds; D010450:Endopeptidases; C091939:ubiquitin isopeptidase; D019759:Carbon-Nitrogen Lyases; C034300:isopeptidase; C038904:zinc fluoride; D005459:Fluorides