Role of EF-hand motif in the activation of neutral trehalase

Miroslava Kopecka^1,2, Dalibor Kosek^1,3, Zdenek Kukacka^3,4, Lenka Rezabkova^1,3, Petr Man^3,4, Petr Novak^3,4_, Tomas Obsil^1,3, Veronika Obsilova¹

¹Institute of Physiology AS CR, v.v.i., Videnska 1083, 142 20 Prague, Czech Republic

² 2^nd Faculty of Medicine, V Uvalu 84, 150 06 Prague, Czech Republic

³Faculty of Science, Charles University in Prague, Albertov 6, 128 43 Prague, Czech Republic
⁴Institute of Microbiology AS CR, v.v.i., Videnska 1083, 142 20 Prague, Czech Republic kopeckamirka@tiscali.cz

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 which 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 (SAXS). 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.

This work was supported by the Czech Science Foundation (Project P207/11/0455) and by Grant Agency of Charles University (Grant 644313).