Role of the EF-hand-like Motif in the 14-3-3 Protein-mediated Activation of Yeast Neutral Trehalase Nth1

• Published in: The Journal of biological chemistry Vol. 289; no. 20; pp. 13948 - 13961
• Main Authors: Kopecka, Miroslava, Kosek, Dalibor, Kukacka, Zdenek, Rezabkova, Lenka, Man, Petr, Novak, Petr, Obsil, Tomas, Obsilova, Veronika
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 16.05.2014; American Society for Biochemistry and Molecular Biology
• Subjects: 14-3-3 Proteins - chemistry; 14-3-3 Proteins - metabolism; 14–3-3; Amino Acid Sequence; Bmh; Calcium; Calcium - metabolism; Catalytic Domain; EF Hand Motifs; Enzyme Activation; Enzyme Mechanisms; H/D Exchange; Mass Spectrometry (MS); Models, Molecular; Neutral Trehalase; Protein Cross-linking; Protein Structure; Protein Structure and Folding; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - metabolism; SAXS; Trehalase - chemistry; Trehalase - metabolism; Enzyme Mechanisms; H/D Exchange; Calcium; 14–3-3; Neutral Trehalase; Mass Spectrometry (MS); Protein Structure; Bmh; Protein Cross-linking; SAXS
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.544551

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. The yeast neutral trehalase Nth1 is activated by the 14-3-3 protein binding. The 14-3-3 protein induces a structural rearrangement of Nth1 with changes within the EF-hand like motif being essential for the activation process The EF-hand-like motif-containing domain is crucial for the 14-3-3-dependent activation of Nth1. Structural basis of the mechanism of Nth1 activation.