Covalent targeting of PP2A scaffold subunit PPP2R1A by Pseudolarolide B enhances holoenzyme assembly to exert anti-inflammatory activity.

Pseudolarolide B (PB), a spirocyclic triterpenoid lactone derived from Pseudolarix amabilis (Nelson) Rehder, demonstrates significant anti-inflammatory properties. However, the precise molecular mechanism underlying this activity has remained elusive. Here, using activity-based protein profiling (ABPP), we identified the scaffolding subunit A (PPP2R1A) of protein phosphatase 2 A (PP2A) as the direct covalent target of PB. We further elucidated that PB covalently binds to Cys317 of PPP2R1A. This modification enhances the structural stability of the A subunit and promotes its capacity to recruit both the catalytic (C) and regulatory (B) subunits, thereby facilitating the assembly of the active PP2A holoenzyme and augmenting its phosphatase activity. Consequently, the enhanced PP2A activity led to the dephosphorylation of key proteins in critical inflammatory signaling pathways. Molecular dynamics simulations provided structural insights, confirming that PB-induced covalent modification stabilizes PPP2R1A and strengthens its interaction with the catalytic subunit. Collectively, our study delineates a novel mechanism whereby PB exerts its anti-inflammatory effects by allosterically promoting PP2A holoenzyme assembly and activity through targeting its scaffolding subunit, offering a new strategy for the pharmacological modulation of PP2 A.