14-3-3 and PDZ domains are important classes of adaptor domains that bind to phosphorylated or non-phosphorylated versions of their target peptides. Peptide binding may in effect be switched on/off via phosphorylation which is thus an interesting mechanism to control protein-protein interactions. As it is often not possible to study binding of both peptide variants in experiment, we tested here the ability of molecular dynamics (MD) simulations to characterize the differential energetic and structural effects of phosphorylation on peptide binding. Binding partners of 14-3-3η domains usually contain a phosphorylated Ser or Thr residue at their binding interface. Binding of unphosphorylated peptides is typically strongly disfavored. Plain MD simulations of 14-3-3η-RSRSTSTP complexes showed that the peptide's phosphate group is ideally coordinated in the binding pocket whereas the respective unphosphorylated side-chain is not. This was consistent with the results of alchemical free energy calculations and explains the binding preference of 14-3-3η for phosphorylated peptides. In contrast, PDZ domains often have a slight preference for non-phosphorylated peptides. They usually bind to the loose C-terminal ends of their partner proteins by coordinating the carboxylate tail of the peptide and via additional hydrogen bonds donated by main chain atoms. Here, we performed MD simulations of EQVSAV and EQVEAV peptides bound to wild-type and mutant versions of the hPTP1E-PDZ2 and MAGI1-PDZ1 domains. The simulations confirmed the previously reported relevance of carboxylate coordination and main chain hydrogen bonds. Both domains coordinated the EQVSAV peptide in a similar manner irrespective of mutations at positions 79 or 85, respectively. In contrast, the presence of Arg79/85 played a prominent role for the EQVEAV peptide. MAGI1-PDZ1 that is lacking Arg, hence showed a looser coordination than hPTP1E-PDZ2. Alchemical simulations for unphosphorylated peptides reproduced well the binding free energy difference of the glutamate mutant. Yet, the binding free energy difference between phosphorylated and non-phosphorylated peptides consistently came out with the wrong sign (-1 instead of +1 kcal/mol), reflecting minor inaccuracies of the CHARMM36m and TIP3P force fields. Overall, MD simulations were found helpful to contrast the conformational stability of phosphorylated vs. non-phosphorylated peptides bound to 14-3-3 and PDZ domains.

Phosphorylation effects on the binding of peptides to PDZ and 14-3-3 domains studied by molecular dynamics simulations

ACS Spring 2022

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