期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 289, 期 20, 页码 13948-13961出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M113.544551
关键词
Calcium; Enzyme Mechanisms; Mass Spectrometry (MS); Protein Cross-linking; Protein Structure; 14-3-3; Bmh; H; D Exchange; SAXS; Neutral Trehalase
资金
- Czech Science Foundation [P207/11/0455]
- Grant Agency of Charles University [644313, 800413]
- Academy of Sciences of the Czech Republic Research Project of the Institute of Physiology [RVO: 67985823]
- Academy of Sciences of the Czech Republic Research Project of the Institute of Microbiology [RVO: 61388971]
- Academy of Sciences of the Czech Republic [OPPK CZ.2.16/3.1.00/24023]
Background: The yeast neutral trehalase Nth1 is activated by the 14-3-3 protein binding. Results: The 14-3-3 protein induces a structural rearrangement of Nth1 with changes within the EF-hand like motif being essential for the activation process. Conclusion: The EF-hand-like motif-containing domain is crucial for the 14-3-3-dependent activation of Nth1. Significance: Structural basis of the mechanism of Nth1 activation. 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 Nth114-3-3 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 Nth114-3-3 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.
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