Abstract
Purpose: :
Guanylyl cyclase activating protein 1 (GCAP1), N-myristoylated neuronal calcium sensor protein, regulates retinal guanylyl cyclase (RetGC) in response to light-dependent changes in free calcium concentrations in photoreceptors. We studied how the interactions between myristoyl group and protein surrounding affected functional properties of GCAP1.
Methods: :
Mutations were introduced to the hydrophobic pocket formed by the parts of EF-hand 1 and 2 and the C-terminal alpha-helix in order to modulate interactions of the N-myristoyl residue with the surrounding amino acid side chains. We tested the stoichiometry of Ca2+ binding, Ca2+-sensitive activation of RetGC1, and the intrinsic tryptophan fluorescence of the mutated GCAP1 in the presence and in the absence of the N-myristoyl group. The position of the myristoyl moiety inside the hydrophobic pocket of GCAP1 between different functional states was verified by NMR-spectroscopy.
Results: :
The absence of myristoyl moiety did not unfold GCAP1, but decreased Ca2+ sensitivity of GCAP1, its apparent affinity for RetGC, and the maximal level of cyclase activation. The myristoyl residue surrouned by EF hands 1 and 2 critically affected Ca2+ binding in the opposite part of the molecule formed by EF hands 3 and 4. According to NMR spectra, myristoyl remained constrained inside the EF1/EF2 semi-globule both in the Ca2+-bound (RetGC inhibitor) and Mg2+-bound (RetGC activator) states in L80F/L176F/V180F mutant. This mutant displayed much higher than wild type affinity for the cyclase, but much lower than the wild type sensitivity to Ca2+. The Phe176 drastically increased the apparent affinity of GCAP1 for the cyclase and also altered Ca2+-sensitivity of the RetGC regulation by shifting it to a higher than normal range of free Ca2+. In comparison with mutations at the mid-portion of the fatty acyl group, substitutions at the bottom and the top of the hydrophobic cavity affected the regulatory properties of GCAP1 to a lesser extent.
Conclusions: :
The fatty acyl group in GCAP1, through its interaction with the C-terminal helix that bridges the N- and the C-proximal portions of the molecule, creates a dynamic tug that tunes GCAP1 into the optimal conformation as a cyclase regulator, by balancing the effectiveness of the interaction with the target enzyme with the optimal Ca2+ sensor function.
Keywords: calcium • protein structure/function • photoreceptors