Systematic optimization and characterization of the MidTAC platform for targeted protein degradation.

Biological proteolysis-targeting chimeras (BioPROTACs), which employ peptide or protein-based targeting ligands to recruit endogenous degradation machinery, offer a promising strategy to expand the scope of targeted protein degradation. The recently discovered Midnolin employs a distinct, ubiquitin-independent "capture-degradation" mechanism that engages the proteasome directly, offering a complementary mechanistic strategy. However, the design principles, domain requirements, and substrate selectivity of Midnolin-based targeting chimeras (MidTAC) remain poorly understood. In this study, we systematically optimized the MidTAC platform and defined its functional boundaries. We demonstrated that optimal degradation activity requires the targeting module, the anti-GFP nanobody VHHGFP4 (variable domain of heavy chain of heavy-chain-only antibody against GFP variant 4), to be fused to the N-terminus of full-length Midnolin, as C-terminal or internal insertions are ineffective. Domain dissection revealed that neither the ubiquitin-like (UBL) domain nor the alpha-helical C-terminal (αHelix-C) domain alone, nor their simple tandem, is sufficient for degradation; the intact spatial conformation of the Midnolin scaffold is essential. Notably, MidTAC efficiently degraded substrate proteins irrespective of their subcellular localization, demonstrating its broad-spectrum utility. Furthermore, fusing the VHL E3 ligase to the C-terminus of Midnolin significantly enhanced degradation efficiency, suggesting a synergistic effect between ubiquitin-independent capture and ubiquitin-dependent recruitment. Collectively, these findings provide the first systematic characterization of MidTAC configuration, domain requirements, and broad-spectrum degradability, establishing a refined framework for engineering of next-generation BioPROTACs with an expanded substrate range.