Structural insights into interactions between ubiquitin specific protease 5 and its polyubiquitin substrates by mass spectrometry and ion mobility spectrometry |
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Authors: | Daniel Scott Robert Layfield Neil J Oldham |
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Institution: | 1.School of Chemistry, University of Nottingham, University Park, Nottingham, NG7 2RD, United Kingdom;2.School of Life Sciences, Queen''s Medical Centre, University of Nottingham, Nottingham, NG7 2UH, United Kingdom |
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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. |
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Keywords: | ubiquitin specific protease 5 deubiquitinase native electrospray ionization-mass spectrometry traveling wave ion mobility spectrometry noncovalent interactions structural proteomics |
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