title: Structural insights into interactions between ubiquitin specific protease 5 and its polyubiquitin substrates by mass spectrometry and ion mobility spectrometry.
abstract: Nanoelectrospray ionization-mass spectrometry and ion mobility-mass spectrometry have been used to study the interactions of the large, multidomain, and conformationally flexible deubiquitinating enzyme ubiquitin specific protease 5 (USP5) with mono- and poly-ubiquitin (Ub) substrates. Employing a C335A active site mutant, mass spectrometry was able to detect the stable and cooperative binding of two mono-Ub molecules at the Zinc-finger ubiquitin binding protein (ZnF-UBP) and catalytic site domains of USP5. Tetra-ubiquitin, in contrast, bound to USP5 with a stoichiometry of 1 : 1, and formed additional interactions with USP5's two ubiquitin associated domains (UBAs). Charge-state distribution and ion mobility analysis revealed that both mono- and tetra-ubiquitin bound to the compact conformation of USP5 only, and that tetra-ubiquitin binding was able to shift the conformational distribution of USP5 from a mixture of extended and compact forms to a completely compact conformation.
authors.affiliation: School of Chemistry, University of Nottingham, University Park, Nottingham, NG7 2RD, United Kingdom.|School of Life Sciences, Queen's Medical Centre, University of Nottingham, Nottingham, NG7 2UH, United Kingdom.
authors.forename: Daniel
authors.initials: D
authors.lastname: Scott
authors.affiliation: School of Life Sciences, Queen's Medical Centre, University of Nottingham, Nottingham, NG7 2UH, United Kingdom.
authors.forename: Robert
authors.initials: R
authors.lastname: Layfield
authors.affiliation: School of Chemistry, University of Nottingham, University Park, Nottingham, NG7 2RD, United Kingdom.
authors.forename: Neil J
authors.initials: NJ
authors.lastname: Oldham
mesh_terms: D001665:Binding Sites; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D025821:Polyubiquitin / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping / Q000379:methods; D060066:Protein Interaction Maps; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism
chemical_list: D025801:Ubiquitin; D025821:Polyubiquitin; D010450:Endopeptidases; C091939:ubiquitin isopeptidase
keywords: deubiquitinase; native electrospray ionization-mass spectrometry; noncovalent interactions; structural proteomics; traveling wave ion mobility spectrometry; ubiquitin specific protease 5