STRUCTURAL STUDY OF THE YEAST ENZYME NEUTRAL TREHALASE AND ITS COMPLEX WITH THE 14-3-3 PROTEIN 

Miroslava Kopecka1,2, Zdenek Kukacka3, Dana Kalabova1, Petr Man3, Tomas Obsil1 and Veronika Obsilova1 

1Institute of Physiology AS CR, v.v.i., Videnska 1083, 14220 Prague, Czech Republic

22nd Faculty of Medicine, Charles University in Prague, V Uvalu 84, 150 06 Prague, Czech Republic

3Institute of Microbiology AS CR, v.v.i., Videnska 1083, 14220 Prague, Czech Republic

miroslava.kopecka@fgu.cas.cz

 

The yeast enzyme neutral trehalase (Nth1, EC 3.2.1.28) from Saccharomyces cerevisiae hydrolyses the non-reducing disaccharide trehalose which serves as an energy source and a universal stress protectant in many different organisms. Enzymatic activity of Nth1 is enhanced by 14-3-3 protein binding in a phosphorylation-dependent manner. Nth1 activity is also regulated by Ca2+ binding to the EF-hand-like motif containing domain of Nth1 [1].

The native TBE PAGE and analytical ultracentrifugation show that Nth1 forms very stable complexes with yeast 14-3-3 proteins [1]. To study the structure of Nth1 alone and its complex with the 14-3-3 protein we used circular dichroism, H/D exchange coupled to mass spectrometry, chemical cross-linking [2] and small angle X-ray scattering (SAXS) [3]. At the same time protein crystallography of Nth1 alone and its complex with 14-3-3 protein is performed.

The low resolution structure of Nth1:14-3-3 protein complex revealed that 14-3-3 protein binding induces a rearrangement of the whole Nth1 molecule and that the region containing the EF-hand-like motif forms a separate domain which interacts with both 14-3-3 protein and catalytic domain of Nth1. We proved that integrity of the EF-hand-like motif is crucial for the 14-3-3 protein mediated activation of Nth1 and for the Ca2+ binding. Our data suggest that the EF-hand-like motif functions as the intermediary through which 14-3-3 protein modulates the function of the catalytic domain of Nth1. These structural changes probably enable the substrate entry into the enzyme active site [3].

Our study of 14-3-3 protein complex with the fully active enzyme Nth1 offers a unique structural view of Nth1 activation enabling us also to better understand the role of the 14-3-3 proteins in regulation of other enzymes.

 

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

 

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2. E. Macakova, M. Kopecka, Z. Kukacka, D. Veisova, P. Novak, P. Man, T. Obsil, V. Obsilova, Biochim. Biophys. Acta. 1830, (2013), 4491 4499.

3. M. Kopecka, D. Kosek, Z. Kukacka, L. Rezabkova, P. Man, P. Novak, T. Obsil, V. Obsilova, J. Biol. Chem. 289, (2014), 13948 13961.