Open AccessTwo ZnF-UBP Domains in Isopeptidase T (USP5)
Author(s) -
G.V. Avvakumov,
John R. Walker,
Song Xue,
Abdellah Allali-Hassani,
Abdalin E. Asinas,
Usha Nair,
Xianyang Fang,
Xiaobing Zuo,
YunXing Wang,
Keith D. Wilkinson,
Sirano DhePaga
Publication year - 2012
Publication title -
biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.43
H-Index - 253
eISSN - 1520-4995
pISSN - 0006-2960
DOI - 10.1021/bi200854q
Subject(s) - ubiquitin , chemistry , residue (chemistry) , enzyme , peptide , allosteric regulation , substrate (aquarium) , cysteine , active site , deubiquitinating enzyme , biochemistry , hydrolase , hydrolysis , stereochemistry , microbiology and biotechnology , biology , ecology , gene
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.