Two ZnF-UBP Domains in Isopeptidase T (USP5)

• Type of Material: Article
• Authors: George V Avvakumov, University of Toronto; John R Walker, University of Toronto; Sheng Xue, University of Toronto; Abdellah Allali-Hassani, University of Toronto; Abdalin Asinas, University of Toronto; Usha B Nair, University of Toronto; Xianyang Fang, National Cancer Institute – Frederick; Xiaobing Zuo, Argonne National Laboratory; Yun-Xing Wane, National Cancer Institute – Frederick; Keith Wilkinson, Emory University; Sirano Dhe-Paganon, University of Toronto
• Language: English
• Date: 2012-02-14
• Publisher: AMER CHEMICAL SOC
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2012 American Chemical Society
• Final Published Version (URL): http://dx.doi.org/10.1021/bi200854q
• Title of Journal or Parent Work: BIOCHEMISTRY
• Volume: 51
• Issue: 6
• Start Page: 1188
• End Page: 1198
• Grant/Funding Information: The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & Co., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research, and the Wellcome Trust. Y-X Wang and X. Fang are supported by the Intramural Research funds of the National Cancer Institute, National Institutes of Health, Department of the Human Health Service, USA.
• Keywords: RESOLUTION; PROTEINS; Science & Technology; RECOGNITION; SOLUTION SCATTERING; BIOLOGICAL MACROMOLECULES; UBIQUITIN; SYSTEM; Life Sciences & Biomedicine; Biochemistry & Molecular Biology

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. © 2012 American Chemical Society.