Structural insights into interactions between ubiquitin specific protease 5 and its polyubiquitin substrates by mass spectrometry and ion mobility spectrometry.
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.
Mesh Terms:
Binding Sites, Endopeptidases, Humans, Models, Molecular, Polyubiquitin, Protein Binding, Protein Conformation, Protein Interaction Mapping, Protein Interaction Maps, Spectrometry, Mass, Electrospray Ionization, Ubiquitin
Binding Sites, Endopeptidases, Humans, Models, Molecular, Polyubiquitin, Protein Binding, Protein Conformation, Protein Interaction Mapping, Protein Interaction Maps, Spectrometry, Mass, Electrospray Ionization, Ubiquitin
Protein Sci.
Date: Aug. 01, 2015
PubMed ID: 25970461
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