Structural model of PP2A-B55α holoenzyme in complex with Parkin Ubl domain
Abstract
Structural models of the quaternary complex comprising the heterotrimeric PP2A holoenzyme and the phosphorylated Parkin Ubl domain were generated using AlphaFold3. The modeled protein assembly included the catalytic subunit Cα (PP2Ac, Uniprot: P67775), the scaffolding subunit PR65A (residues 1–589, Uniprot: P30153), the regulatory subunit B55α (residues 1–447, Uniprot: P63151-1), the phosphorylated Parkin Ubl domain (Uniprot: O60260), and two Mn2+ ions. To determine the optimal substrate boundary, we systematically varied Parkin construct lengths, including the minimal Ubl domain (residues 1–78), extended linker (residues 1–99), and full-length Parkin (residues 1–465). Inclusion of full-length Parkin or extended disordered linker—where Ile142 in the RING0 domain is the first downstream structured residue—introduced excessive conformational entropy and steric noise during diffusion sampling, steering the substrate domain away from the catalytic cleft. Defining the substrate construct as Parkin residues 1–88 preserved the folded Ubl core (residues 1–71) and the functional basic C-terminal tail (residues 72–88) while eliminating entropic sampling penalties. Phosphorylation at Ser65 and the presence of the active-site binuclear metal center were explicitly defined; control prediction runs omitting either the metal cofactors or the phosphoryl group failed to position the substrate near the active site. Structure generation was performed across five independent runs using distinct random seeds (yielding 5 models per run; 25 models in total). Models were evaluated based on the interface predicted TM-score (ipTM), inter-subunit Predicted Aligned Error (PAE) matrices between B55α and the Ubl domain, and catalytic geometry (defined by spatial proximity between the pSer65 phosphoryl group and the binuclear catalytic metal center). Across all calculations, 15 out of 25 models (60%) converged on a consistent quaternary architecture. Representative Model is uploaded.
