Role of the EF-hand-like motif in the 14-3-3 protein-mediated activation of yeast neutral trehalase Nth1.

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 EF-hand-like motif, whose role in the activation of Nth1 is unclear. In this work, the structure of the Nth1·14-3-3 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 Nth1·14-3-3 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 EF-hand like motif-containing domain functions as the intermediary through which the 14-3-3 protein modulates the function of the catalytic domain of Nth1.

Structural basis of the 14-3-3 protein-dependent activation of yeast neutral trehalase Nth1.

BACKGROUND: Trehalases are highly conserved enzymes catalyzing the hydrolysis of trehalose in a wide range of organisms. The activity of yeast neutral trehalase Nth1 is regulated in a 14-3-3- and a calcium-dependent manner. The Bmh proteins (the yeast 14-3-3 isoforms) recognize phosphorylated Nth1 and enhance its enzymatic activity through an unknown mechanism. METHODS: To investigate the structural basis of interaction between Nth1 and Bmh1, we used hydrogen/deuterium exchange coupled to mass spectrometry, circular dichroism spectroscopy and homology modeling to identify structural changes occurring upon the complex formation. RESULTS: Our results show that the Bmh1 protein binding affects structural properties of several regions of phosphorylated Nth1: the N-terminal segment containing phosphorylation sites responsible for Nth1 binding to Bmh, the region containing the calcium binding domain, and segments surrounding the active site of the catalytic trehalase domain. The complex formation between Bmh1 and phosphorylated Nth1, however, is not accompanied by the change in the secondary structure composition but rather the change in the tertiary structure. CONCLUSIONS: The 14-3-3 protein-dependent activation of Nth1 is based on the structural change of both the calcium binding domain and the catalytic trehalase domain. These changes likely increase the accessibility of the active site, thus resulting in Nth1 activation. GENERAL SIGNIFICANCE: The results presented here provide a structural view of the 14-3-3 protein-dependent activation of yeast neutral trehalase Nth1, which might be relevant to understand the process of Nth1 activity regulation as well as the role of the 14-3-3 proteins in the regulation of other enzymes.