Rajanikanth Vangipurapu, Sharma Anand Kumar, Rajyalakshmi Meduri, Chandra Kousik, Chary Kandala V R, Sharma Yogendra
CSIR-Centre for Cellular and Molecular Biology (CCMB), Hyderabad 500007, India.
Biochemistry. 2015 Feb 3;54(4):1111-22. doi: 10.1021/bi501134g. Epub 2015 Jan 21.
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 Ca(2+) binding ability of the disabled EF-1 loop by appropriate mutations. The concurrence of canonical EF-1 and N-myristoylation considerably decreased the overall Ca(2+) affinity, conformational flexibility, and functional activation of downstream effecter molecules (i.e., PI4Kβ). Of a particular note, Ca(2+) induced conformational change (which is the first premise for a CaBP to be considered as sensor) is considerably reduced in myristoylated proteins in which Ca(2+)-binding to EF-1 is restored. Moreover, Ca(2+), 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 Ca(2+) 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 Ca(2+) specific signal transduction to downstream effecter molecules despite Ca(2+) binding. Thus, the EF-1 disability is a prerequisite in order to append myristoylation signaling while preserving structural robustness and Ca(2+) sensitivity/specificity in NCS-1.
神经元钙传感器（NCS）蛋白家族的许多成员显著共存两种特征，即N-肉豆蔻酰化和隐蔽的EF-1基序。我们通过适当的突变恢复失活的EF-1环的Ca(2+)结合能力，研究了神经元钙传感器-1（NCS-1）中这种相关性背后的原理。经典EF-1和N-肉豆蔻酰化的同时存在显著降低了下游效应分子（即PI4Kβ）的整体Ca(2+)亲和力、构象灵活性和功能激活。特别值得注意的是，在恢复了Ca(2+)与EF-1结合的肉豆蔻酰化蛋白中，Ca(2+)诱导的构象变化（这是CaBP被视为传感器的首要前提）显著降低。此外，Ca(2+)原本会增强PI4Kβ的酶活性（由NCS-1调节），但在具有经典EF-1的肉豆蔻酰化突变体中，Ca(2+)会导致被调节的PI4Kβ活性进一步下降，这表明在结构和功能水平上Ca(2+)信号和特异性丧失。这项研究证实了NCS-1中肉豆蔻酰化与隐蔽EF-1之间存在紧密联系。打破这种联系会导致尽管Ca(2+)结合，但Ca(2+)特异性信号无法转导至下游效应分子。因此，EF-1失活是在NCS-1中附加肉豆蔻酰化信号同时保持结构稳健性和Ca(2+)敏感性/特异性的先决条件。
