Role of the EF-hand-like motif in the 14-3-3 protein-mediated activation of yeast neutral trehalase Nth1.
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.
From the Institute of Physiology and the Second Faculty of Medicine, Charles University, V Uvalu 84, 150 06 Prague, Czech Republic, and.
Miroslava
M
Kopecka
From the Institute of Physiology and the Departments of Physical and Macromolecular Chemistry and.
Dalibor
D
Kosek
the Institute of Microbiology, Academy of Sciences of the Czech Republic v.v.i., Videnska 1083, 14220 Prague, Czech Republic, Biochemistry, Faculty of Science, Charles University, Hlavova 2030, 12843 Prague, Czech Republic.
Zdenek
Z
Kukacka
From the Institute of Physiology and the Departments of Physical and Macromolecular Chemistry and.
Lenka
L
Rezabkova
the Institute of Microbiology, Academy of Sciences of the Czech Republic v.v.i., Videnska 1083, 14220 Prague, Czech Republic, Biochemistry, Faculty of Science, Charles University, Hlavova 2030, 12843 Prague, Czech Republic.
Petr
P
Man
the Institute of Microbiology, Academy of Sciences of the Czech Republic v.v.i., Videnska 1083, 14220 Prague, Czech Republic, Biochemistry, Faculty of Science, Charles University, Hlavova 2030, 12843 Prague, Czech Republic.
Petr
P
Novak
From the Institute of Physiology and the Departments of Physical and Macromolecular Chemistry and.
Tomas
T
Obsil
From the Institute of Physiology and obsilova@biomed.cas.cz.
Veronika
V
Obsilova
D048948:14-3-3 Proteins / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D020134:Catalytic Domain; D020832:EF Hand Motifs; D004789:Enzyme Activation; D008958:Models, Molecular; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*; D014198:Trehalase / Q000737:chemistry / Q000378:metabolism*
D048948:14-3-3 Proteins; C075852:BMH1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014198:Trehalase; D002118:Calcium
14–3-3; Bmh; Calcium; Enzyme Mechanisms; H/D Exchange; Mass Spectrometry (MS); Neutral Trehalase; Protein Cross-linking; Protein Structure; SAXS