Gli3/Spop Multivalent Interactions are Concentration-Dependent

Abstract

It has been recently shown that many ubiquitin ligase substrates have multiple weak degrons instead of one strong one. These substrates are largely disordered, which allows for access to each degron. The biological function of these multiple degrons is unclear. Gli3, an intrinsically disordered, zinc-finger-containing transcription factor, is an effector of Hedgehog (Hh) signaling. Gli3 degradation is mediated by the E3 cullin-RING ubiquitin ligase speckle-type POZ protein (SPOP). SPOP binding motifs within Gli3 were identified by peptide microarray analysis and Gli3 was found to potentially have >70 binding motifs. Each SPOP monomer binds a single SBC motif, implying the abundance of motifs serves a function other than stoichiometric binding. SPOP itself is capable of forming concentration-dependent, dynamic oligomeric complexes. In this study, biophysical and structural techniques were used to characterize the oligomeric properties of SPOP and how binding of a multivalent Gli3 substrate affects these properties. Preliminary results suggest that individual degrons have similar weak binding affinities, with no site preferentially bound. Results also show that there is a concentration threshold for interactions between Gli3 and SPOP to occur. Further studies will continue to investigate this concentration-dependence and assess how this mechanism of binding may regulate Gli3 function and correct Hh signal transduction.