Role of the EF-hand-like Motif in the 14-3-3 Protein-mediated Activation of Yeast Neutral Trehalase Nth1
Abstract
Trehalases hydrolyze the non-reducing disaccharide trehalose amassed by cells as a universal protectant and storage carbohydrate. Recently, it has been shown that the activity of neutral trehalase Nth1 from Saccharomyces cerevisiae is mediated by the 14-3-3 protein binding that modulates the structure of both the catalytic domain and the region containing the EFhand-like motif, whose role in the activation of Nth1 is unclear.
In this work, the structure of the Nth114-3-3 complex and the importance of the EF-hand-like motif were investigated using site-directed mutagenesis, hydrogen/deuterium exchange coupled to mass spectrometry, chemical cross-linking, and small angle x-ray scattering. The low resolution structural views of Nth1 alone and the Nth114-3-3 complex show that the 14-3-3 protein binding induces a significant structural rearrangement of the whole Nth1 molecule.
The EF-hand-like motif containing region forms a separate domain that interacts with both the 14-3-3 protein and the catalytic trehalase domain. The structural integrity of the EF-hand like motif is essential for the 14-3-3 protein-mediated activation of Nth1, and calcium binding, although not required for the activation, facilitates this process by affecting its structure.
Our data suggest that the EFhand like motif-containing domain functions as the intermediary through which the 14-3-3 protein modulates the function of the catalytic domain of Nth1.