Probing Affinity and Ubiquitin Linkage Selectivity of Ubiquitin-Binding Domains Using Mass Spectrometry

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• 作者：Kleitos Sokratous ; Lucy V. Roach ; Debora Channing ; Joanna Strachan ; Jed Long ; Mark S. Searle ; Robert Layfield ; Neil J. Oldham
• 刊名：The Journal of the American Chemical Society
• 出版年：2012
• 出版时间：April 11, 2012
• 年：2012
• 卷：134
• 期：14
• 页码：6416-6424
• 全文大小：381K
• 年卷期：v.134,no.14(April 11, 2012)
• ISSN：1520-5126

文摘

Non-covalent interactions between ubiquitin (Ub)-modified substrates and Ub-binding domains (UBDs) are fundamental to signal transduction by Ub receptor proteins. Poly-Ub chains, linked through isopeptide bonds between internal Lys residues and the C-terminus of Ub, can be assembled with varied topologies to mediate different cellular processes. We have developed and applied a rapid and sensitive electrospray ionization-mass spectrometry (ESI-MS) method to determine isopeptide linkage-selectivity and affinity of poly-Ub路UBD interactions. We demonstrate the technique using mono-Ub and poly-Ub complexes with a number of 伪-helical and zinc-finger (ZnF) UBDs from proteins with roles in neurodegenerative diseases and cancer. Affinities in the 2鈥?00 渭M range were determined to be in excellent agreement with data derived from other biophysical techniques, where available. Application of the methodology provided further insights into the poly-Ub linkage specificity of the hHR23A-UBA2 domain, confirming its role in Lys48-linked poly-Ub signaling. The ZnF UBP domain of isopeptidase-T showed no linkage specificity for poly-Ub chains, and the Rabex-5 MIU also exhibited little or no specificity. The discovery that a number of domains are able to bind cyclic Lys48 di-Ub with affinities similar to those for the acyclic form indicates that cyclic poly-Ub may be capable of playing a role in Ub-signaling. Detection of a ternary complex involving Ub interacting simultaneously with two different UBDs demonstrated the co-existence of multi-site interactions, opening the way for the study of crosstalk between individual Ub-signaling pathways.