Capturing the recognition dynamics of para-sulfonato- calix[4]arenes by cytochrome c: towards a quantitative free energy assessment

Calix[n]arenes selective recognition of protein surface covers a broad range of timely applications, notably to control protein assembly and crystallization or trap partially disordered proteins. Here, the interaction of para-sulfonated calix-[4]-arenes with cytochrome c is investigated by all-atom, explicit water molecular dynamics simulations which allow to characterize two binding sites in neat agreement with experimental evidences. Free energy calculations based on the MM-PBSA and the attach-pull-release (APR) methods highlight key residues implicated in the recognition process and provide binding free energy results in quantitative agreement with the isothermal titration calorimetry. Our study emphasizes the role of MD simulations to capture the ``walk" of sulfonated calix-[4]-arenes on cytochrome c surface, with R13 as a pivotal interacting residue. Our MD investigation allows, through the QHMB method, to probe an allosteric reinforcement of several per-residues interactions upon calixarene binding, which suggest a more complex mode of action of these supramolecular auxiliaries.