Recognition of polyubiquitin isoforms by the multiple ubiquitin binding modules of isopeptidase T.
The conjugation of polyubiquitin to target proteins acts as a signal that regulates target stability, localization, and function. Several ubiquitin binding domains have been described, and while much is known about ubiquitin binding to the isolated domains, little is known with regard to how the domains interact with polyubiquitin in the context of full-length proteins. Isopeptidase T (IsoT/USP5) is a deubiquitinating enzyme that is largely responsible for the disassembly of unanchored polyubiquitin in the cell. IsoT has four ubiquitin binding domains: a zinc finger domain (ZnF UBP), which binds the proximal ubiquitin, a UBP domain that forms the active site, and two ubiquitin-associated (UBA) domains whose roles are unknown. Here, we show that the UBA domains are involved in binding two different polyubiquitin isoforms, linear and K48-linked. Using isothermal titration calorimetry, we show that IsoT has at least four ubiquitin binding sites for both polyubiquitin isoforms. The thermodynamics of the interactions reveal that the binding is enthalpy-driven. Mutation of the UBA domains suggests that UBA1 and UBA2 domains of IsoT interact with the third and fourth ubiquitins in both polyubiquitin isoforms, respectively. These data suggest that recognition of the polyubiquitin isoforms by IsoT involves considerable conformational mobility in the polyubiquitin ligand, in the enzyme, or in both.
Reyes-Turcu
Francisca E
FE
Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Shanks
John R
JR
Komander
David
D
Wilkinson
Keith D
KD
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D010450:Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009154:Mutation; D011485:Protein Binding / Q000502:physiology; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination / Q000502:physiology; D016335:Zinc Fingers / Q000502:physiology
D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D010450:Endopeptidases; C091939:ubiquitin isopeptidase