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

J Biol Chem. 2014 May 16;289(20):13948-61. doi: 10.1074/jbc.M113.544551. Epub 2014 Apr 8.


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.

Keywords: 14–3-3; Bmh; Calcium; Enzyme Mechanisms; H/D Exchange; Mass Spectrometry (MS); Neutral Trehalase; Protein Cross-linking; Protein Structure; SAXS.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 14-3-3 Proteins / chemistry
  • 14-3-3 Proteins / metabolism
  • Amino Acid Sequence
  • Calcium / metabolism
  • Catalytic Domain
  • EF Hand Motifs*
  • Enzyme Activation
  • Models, Molecular
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Trehalase / chemistry
  • Trehalase / metabolism*


  • 14-3-3 Proteins
  • BMH1 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • Trehalase
  • Calcium