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Structural insights into interactions between ubiquitin specific protease 5 and its polyubiquitin substrates by mass spectrometry and ion mobility spectrometry
Author(s) -
Scott Daniel,
Layfield Robert,
Oldham Neil J.
Publication year - 2015
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.2692
Subject(s) - deubiquitinating enzyme , chemistry , ubiquitin , electrospray ionization , mass spectrometry , ion mobility spectrometry , protease , enzyme , biochemistry , chromatography , gene
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.