Beyond CRBN and VHL: Parkin driven lysine-based ternary complexes for selective β3-Tubulin degradation

Targeted protein degradation (TPD) is a rapidly emerging and potentially transformative therapeutic modality. However, PROTACs have primarily relied on only two E3 ligases, CRBN and VHL, leaving the vast majority of over 600 human ubiquitin ligases unexplored. In this study, we explored Parkin, an RBR-type E3 ligase, as a new recruiter for targeted protein degradation. Parkin is known to ubiquitinate several cytoskeletal and mitochondrial proteins in vivo. Since Parkin has not yet been structurally incorporated into PROTAC workflows, we examined all available human crystal structures to understand its mechanism of ubiquitin transfer and distinguish between its active and autoinhibited conformations. A full-length, catalytically competent Parkin model with all functional domains was built using AlphaFold 2. Using this active Parkin structure, we generated multiple ternary complex conformations with β3-tubulin employing PROTACs varying in linker length and composition. These ternary complexes were filtered through lysine proximity screening, dynamic conformational sampling, interface analysis, and tubulin-isoform-specific lysine accessibility. Selected ternary complexes were further subjected to comprehensive MD studies and analysed using BSA, PCA, Time-lagged independent component analysis (tICA), and Markov state modeling (MSM), revealing stable, degradation-competent conformations. This study paves the way for leveraging Parkin as a versatile E3 ligase in targeted protein degradation, offering new opportunities to modulate proteins implicated in critical cellular processes and complex pathophysiological conditions.