Mechanistic insights into macromolecular recognition at the PP2A-B55α regulatory groove from structure-based modeling and molecular dynamics.
Protein phosphatase 2 A (PP2A) achieves signaling specificity through regulatory B subunits, but the chemical and structural determinants of regulatory-subunit recognition surfaces remain incompletely defined. The first PP2A-B55α complex structure identified a regulatory groove on the β-propeller surface, spatially separated from the catalytic site and occupied by a FAM122A regulatory segment. This groove therefore represents a macromolecular recognition surface that can be systematically probed for cyclic peptide engagement. Here, 8466 cyclic peptides from CycPeptMPDB were screened against the B55α regulatory groove, and KarmaDock score-based ranking prioritized seven representative cyclic peptides for detailed structural and energetic analysis. Refined simulations showed peptide-dependent modulation of conformational stability, convergence to stable bound states, selective stabilization of the regulatory groove, and retained flexibility of the extended A-subunit arm. Triplicate and extended simulations of P-659, together with triplicate simulations of the top candidate P-589, further supported reproducible structural behavior and binding energetics. Persistent hydrogen-bonding patterns suggested peptide-specific anchoring through Asp190, Asp197, Asp333, Tyr330, Ser280, and Lys345. Peptide binding was accompanied by the expected displacement of solvent from the solvent-exposed groove interior and localized reorganization of interfacial hydration, while residue-wise thermodynamic profiling identified Lys81, Met215, Glu216, Phe273, Tyr330, Asp333, and Phe336 as key solvent-response residues. Alanine scanning identified Asp197 as the principal energetic hotspot, with ligand-specific contributions from Ser280, Tyr330, and Asp333. Binding free-energy calculations indicated balanced gas-phase and solvation contributions, with P-589 showing the most favorable relative MM-GBSA binding-energy estimate among the analyzed peptides (ΔGTOTAL = -61.84 ± 0.43 kcal/mol). Together, these data establish a computational, structure-, dynamics-, and energetics-resolved framework for cyclic peptide recognition at the PP2A-B55α regulatory groove and define testable hypotheses for experimental validation.