A double system in a single box setup was used; here, both the protein and peptide (e.g., ACE-AXA-NH 2 ) are situated at a distance of 3 nm in the same box, which ensures charge neutrality during the alchemical transition 111 . To prevent consequential protein-peptide interactions, a single Cα in each molecule was positionally restrained. We used the CHARMM36m 112 (with CHARMM-modified TIP3P 113 ) and Amber14sb (with TIP3P 114 ) force fields.