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).