Liaison between Myristoylation and Cryptic EF-Hand Motif Confers Ca²⁺ Sensitivity to Neuronal Calcium Sensor‑1

Many members of the neuronal calcium sensor (NCS) protein family have a striking coexistence of two characteristics, that is, N-myristoylation and the cryptic EF-1 motif. We investigated the rationale behind this correlation in neuronal calcium sensor-1 (NCS-1) by restoring Ca²⁺ binding ability of the disabled EF-1 loop by appropriate mutations. The concurrence of canonical EF-1 and N-myristoylation considerably decreased the overall Ca²⁺ affinity, conformational flexibility, and functional activation of downstream effecter molecules (i.e., PI4Kβ). Of a particular note, Ca²⁺ induced conformational change (which is the first premise for a CaBP to be considered as sensor) is considerably reduced in myristoylated proteins in which Ca²⁺-binding to EF-1 is restored. Moreover, Ca²⁺, which otherwise augments the enzymatic activity of PI4Kβ (modulated by NCS-1), leads to a further decline in the modulated PI4Kβ activity by myristoylated mutants (with canonical EF-1) pointing toward a loss of Ca²⁺ signaling and specificity at the structural as well as functional levels. This study establishes the presence of the strong liaison between myristoylation and cryptic EF-1 in NCS-1. Breaking this liaison results in the failure of Ca²⁺ specific signal transduction to downstream effecter molecules despite Ca²⁺ binding. Thus, the EF-1 disability is a prerequisite in order to append myristoylation signaling while preserving structural robustness and Ca²⁺ sensitivity/specificity in NCS-1.