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Probing Affinity and Ubiquitin Linkage Selectivity of Ubiquitin-Binding Domains Using Mass Spectrometry
journal contribution
posted on 2016-02-21, 14:52 authored by Kleitos Sokratous, Lucy V. Roach, Debora Channing, Joanna Strachan, Jed Long, Mark S. Searle, Robert Layfield, Neil J. OldhamNon-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.