Biophysical Characterization of the Multivalent Gli3/SPOP Interactions

Abstract

Contrary to traditional protein binding mechanisms, many ubiquitin ligase substrates have multiple weak degrons instead of one strong one. These proteins are largely disordered, allowing 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 SPOP (speckle-type POZ protein). Ser/Thr rich SPOP-binding consensus (SBC) motifs were identified as interaction sites for SPOP. Based upon sequence analysis, Gli3 contains 13 of these motifs. Each SPOP monomer binds a single SBC motif, implying the abundance of motifs serves a function other than stoichiometric binding. This suggests several models for SPOP/Gli3 binding: (1) a dynamic complex, wherein single SPOP oligomers interact with single Gli3s in fast equilibrium; (2) a static multi-valent complex wherein each Gli3 has multiple SPOP binding partners; or (3) a high-order oligomeric complex wherein multiple Gli3 molecules interact with multiple SPOP oligomers, assembling into large complexes that can lead to phase transitions. In this study, biophysical techniques were used to probe the function of motifs in the regulation of SPOP binding. Preliminary results suggest that individual degrons have similar weak binding affinities, with no site preferentially bound. These binding sites may also play a function in the regulation of Gli3 degradation in vivo. Gli3 function as an activator or a repressor is determined by cellular Hh availability. In its active form, Gli3 enters the nucleus and, as a labile transcription activator, is rapidly degraded. The function SBC motifs play in this degradation process will be analyzed in vivo using various truncations and deletions of Gli3. Further studies will elucidate the role of multiple dispersed degrons for the temporal and spatial control of Hh signaling.