Yorkie-Warts Complexes are an Ensemble of Interconverting Conformers Formed by Multivalent Interactions

Abstract

pathways. in recent years, in vivo studies have shown that the protein Histidine triad nucleotide-binding protein 1 (HINT1) inhibits the ubiquitylation activity of various SCF ligases that regulate the NF-kB signaling pathway and cell cycle progression by promoting the degradation of substratesIkBa and p27 Kip1 , respectively. However, the mechanism of interaction between HINT1 and SCF ligases and how HINT1 causes their inhibition is not understood. Many questions remain unanswered such as the specific binding partner of HINT1 within the SCF complex and how HINT1 is able to inhibit the activity of unique SCF ligases. in total, the 173 kDa SCF ligase complex consists of a scaffolding protein, Cullin1 (90 kDa), that utilizes Rbx1 (12 kDa) to recruit E2 ubiquitin conjugating enzymes (e.g. UBE2D2) and substrates through the adaptor and substrate receptor proteins, Skp1 (17 kDa) and b-TrCP1 (54 kDa) respectively, to promote substrate ubiquitylation. Through co-elution in gel-filtration chromatography, I have assembled the SCF E3 ligase that ubiquitylates IkBa and have demonstrated that HINT1 (28 kDa) binds to this ligase. I have performed biochemical experiments to measure ubiquitylation combined with HDX-MS and other biophysical experiments to characterize the binding affinity and the specific binding interface of HINT1 on the SCF E3 ligase. The results shed light on the molecular mechanism of inhibition of SCF E3 ligases by HINT1.