Disordered Motifs and Domains in Cell Control
Tuesday Speaker Abstracts
The Role of Multivalent Interactions of Tumor Suppressor SPOP with Gli3 in Regulating
Ubiquitination
Melissa Marzah, Wendy Pierce, Jihun Lee, Amanda Nourse, Suresh Marada, Stacey
Ogden,
Tanja Mittag
.
1
St. Jude Children's Research Hospital, Memphis, TN, USA
Multivalent protein interactions can give rise to avidity effects, ultrasensitivity, phase separation
and spatial organization. The tumor suppressor SPOP, the substrate adaptor of a ubiquitin ligase,
self-associates into large oligomers. We predict that its predominantly intrinsically disordered
substrate Gli3 contains many weak SPOP binding motifs. Multivalency of SPOP and Gli3 for
each other, i.e. the ability of SPOP oligomers to bind many SPOP binding motifs in Gli3 and of
Gli3 molecules to interact with many SPOP monomeric units, suggests a highly concentration-
dependent interaction.
We used a peptide microarray, NMR spectroscopy, fluorescence methods and analytical
ultracentrifugation (AUC) to identify SPOP binding motifs in Gli3. Importantly, even very weak
motifs contributed substantially to ubiquitination demonstrating their functional relevance.
Using AUC, size exclusion chromatography, and static light scattering, we demonstrated that
two distinct dimerization domains in SPOP mediate the formation of concentration-dependent
higher-order SPOP homo-oligomers. Our thermodynamic characterization supports an isodesmic
self-association model, indicating that the size of the oligomers is limited only by protein
availability. Importantly, our full description of SPOP’s valency as a function of concentration
now permits the quantitative characterization of its interactions with multivalent binding
partners.
Does the multivalent Gli3/SPOP interaction give rise to avidity effects in small complexes or to
large cross-linked complexes? We show that Gli3 and SPOP co-localize into nuclear “bodies” in
cells suggesting that these “bodies” may be mediated by (1) specific oligomerization through
defined interfaces in SPOP; and (2) the weak multivalent interactions between Gli3 and SPOP. It
has been suggested that concentration-dependent assembly/disassembly of large, multivalent
complexes provides a mechanism for fine-tuning signaling cascades. Multivalent interactions of
SPOP with its substrates may be an avenue to regulate levels of proteins critical in development
and proliferation in a concentration-dependent manner.
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