Role of myristoylation in modulating PCaP1 interaction with calmodulin

Plasma membrane-associated Cation-binding Protein 1 (PCaP1) belongs to the plant-unique DREPP protein family with largely unknown biological functions but ascertained roles in plant development and calcium (Ca²⁺) signaling. PCaP1 is anchored to the plasma membrane via N-myristoylation and a polybasic cluster, and its N-terminal region can bind Ca²⁺/calmodulin (CaM). However, the molecular determinants of PCaP1-Ca²⁺-CaM interaction and the functional impact of myristoylation in the complex formation and Ca²⁺ sensitivity of CaM remained to be elucidated. Herein, we investigated the direct interaction between Arabidopsis PCaP1 (AtPCaP1) and CaM1 (AtCaM1) using both myristoylated and non-myristoylated peptides corresponding to the N-terminal region of AtPCaP1. ITC analysis showed that AtCaM1 forms a high affinity 1:1 complex with AtPCaP1 peptides and the interaction is strictly Ca²⁺-dependent. Spectroscopic and kinetic Ca²⁺ binding studies showed that the myristoylated peptide dramatically increased the Ca²⁺-binding affinity of AtCaM1 and slowed the Ca²⁺ dissociation rates from both the C- and N-lobes, thus suggesting that the myristoylation modulates the mechanism of AtPCaP1 recognition by AtCaM1. Furthermore, NMR and CD spectroscopy revealed that the structure of both the N- and C-lobes of Ca²⁺-AtCaM1 changes markedly in the presence of the myristoylated AtPCaP1 peptide, which assumes a helical structure in the final complex. Overall, our results indicate that AtPCaP1 biological function is strictly related to the presence of multiple ligands, i.e., the myristoyl moiety, Ca²⁺ ions and AtCaM1 and only a full characterization of their equilibria will allow for a complete molecular understanding of the putative role of PCaP1 as signal protein.