title: Probing affinity and ubiquitin linkage selectivity of ubiquitin-binding domains using mass spectrometry.
abstract: 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·UBD interactions. We demonstrate the technique using mono-Ub and poly-Ub complexes with a number of α-helical and zinc-finger (ZnF) UBDs from proteins with roles in neurodegenerative diseases and cancer. Affinities in the 2-200 μM 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.
authors.affiliation: School of Chemistry, University of Nottingham, University Park NG7 2RD, UK.
authors.forename: Kleitos
authors.initials: K
authors.lastname: Sokratous
authors.forename: Lucy V
authors.initials: LV
authors.lastname: Roach
authors.forename: Debora
authors.initials: D
authors.lastname: Channing
authors.forename: Joanna
authors.initials: J
authors.lastname: Strachan
authors.forename: Jed
authors.initials: J
authors.lastname: Long
authors.forename: Mark S
authors.initials: MS
authors.lastname: Searle
authors.forename: Robert
authors.initials: R
authors.lastname: Layfield
authors.forename: Neil J
authors.initials: NJ
authors.lastname: Oldham
mesh_terms: D001665:Binding Sites; D045744:Cell Line, Tumor; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D015398:Signal Transduction; D012997:Solvents / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D025801:Ubiquitin / Q000737:chemistry*; D014867:Water / Q000737:chemistry; D016335:Zinc Fingers
chemical_list: D010455:Peptides; D011506:Proteins; D012997:Solvents; D025801:Ubiquitin; D014867:Water; D008239:Lysine