Scott Daniel, Layfield Robert, Oldham Neil J
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.
Protein Sci. 2015 Aug;24(8):1257-63. doi: 10.1002/pro.2692. Epub 2015 May 29.
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.
纳米电喷雾电离质谱和离子淌度质谱已被用于研究大型、多结构域且构象灵活的去泛素化酶泛素特异性蛋白酶5（USP5）与单泛素和多聚泛素（Ub）底物之间的相互作用。利用C335A活性位点突变体，质谱能够检测到两个单泛素分子在USP5的锌指泛素结合蛋白（ZnF-UBP）和催化位点结构域处的稳定且协同的结合。相比之下，四聚泛素以1:1的化学计量比与USP5结合，并与USP5的两个泛素相关结构域（UBA）形成额外的相互作用。电荷态分布和离子淌度分析表明，单泛素和四聚泛素均仅与USP5的紧密构象结合，且四聚泛素的结合能够将USP5的构象分布从伸展形式和紧密形式的混合物转变为完全紧密的构象。
