Molecular Insights into the Trapping Effect of Ca²⁺ in Protein Kinase A: A Molecular Dynamics Study

Protein kinase A has become a model system for the study of kinases, and therefore, a comprehensive understanding of the underlying molecular mechanisms in its catalytic cycle is of crucial importance. One of the aspects that has received recent attention is the role that metal cofactors play in the catalytic cycle. Although Mg²⁺ is the well-known physiological ion used by protein kinases, Ca²⁺ ions can also assist the phosphoryl transfer reaction but with lower catalytic activities. This inhibitory effect has been attributed to the ability of Ca²⁺ to trap the reaction products at the active site, and it has been proposed as a possible regulatory mechanism of the enzyme. Thus, in order to get a clearer understanding of these molecular events, computational simulations in the product state of PKA, in the presence of Mg²⁺ and Ca²⁺ ions, were performed through molecular dynamics (MD). Different protonation states of the active site were considered in order to model the different mechanistic pathways that have been proposed. Our results show that different protonation states of the phosphorylated serine residue at the peptide substrate (pSer21), as well as the protonation state of residue Asp166, can have a marked influence on the flexibility of regions surrounding the active site. This is the case of the glycine-rich loop, a structural motif that is directly involved in the release of the products from the PKA active site. MD simulations were capable to reproduce the crystallographic conformations but also showed other conformations not previously reported in the crystal structures that may be involved in enhancing the affinity of pSP20 to PKA in the presence of Ca²⁺. Hydrogen bonding interactions at the PKA-pSP20 interface were influenced whether by the protonation state of the active site or by the metal cofactor used by the enzyme. Altogether, our results provide molecular aspects into the inhibitory mechanism of Ca²⁺ in PKA and suggest which is the most probable protonation state of the phosphorylated product at the active site.