Structural basis of the 14-3-3 protein-dependent activation of yeast neutral trehalase Nth1 |
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Authors: | Eva Macakova Miroslava Kopecka Zdenek Kukacka Dana Veisova Petr Novak Petr Man Tomas Obsil Veronika Obsilova |
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Institution: | 1. Institute of Physiology, Academy of Sciences of the Czech Republic v.v.i., Videnska 1083, 14220 Prague, Czech Republic;2. 2nd Faculty of Medicine, Charles University, V Uvalu 84, 150 06 Prague, Czech Republic;3. Institute of Microbiology, Academy of Sciences of the Czech Republic v.v.i., Videnska 1083, 14220 Prague, Czech Republic;4. Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030, 12843 Prague, Czech Republic;5. Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Hlavova 2030, 12843 Prague, Czech Republic |
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Abstract: | BackgroundTrehalases are highly conserved enzymes catalyzing the hydrolysis of trehalose in a wide range of organisms. The activity of yeast neutral trehalase Nth1 is regulated in a 14-3-3- and a calcium-dependent manner. The Bmh proteins (the yeast 14-3-3 isoforms) recognize phosphorylated Nth1 and enhance its enzymatic activity through an unknown mechanism.MethodsTo investigate the structural basis of interaction between Nth1 and Bmh1, we used hydrogen/deuterium exchange coupled to mass spectrometry, circular dichroism spectroscopy and homology modeling to identify structural changes occurring upon the complex formation.ResultsOur results show that the Bmh1 protein binding affects structural properties of several regions of phosphorylated Nth1: the N-terminal segment containing phosphorylation sites responsible for Nth1 binding to Bmh, the region containing the calcium binding domain, and segments surrounding the active site of the catalytic trehalase domain. The complex formation between Bmh1 and phosphorylated Nth1, however, is not accompanied by the change in the secondary structure composition but rather the change in the tertiary structure.ConclusionsThe 14-3-3 protein-dependent activation of Nth1 is based on the structural change of both the calcium binding domain and the catalytic trehalase domain. These changes likely increase the accessibility of the active site, thus resulting in Nth1 activation.General significanceThe results presented here provide a structural view of the 14-3-3 protein-dependent activation of yeast neutral trehalase Nth1, which might be relevant to understand the process of Nth1 activity regulation as well as the role of the 14-3-3 proteins in the regulation of other enzymes. |
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Keywords: | HDX H/D exchange HDX&ndash MS H/D exchange coupled to mass spectrometry DSS disuccinimidyl suberate DSG disuccinimidyl glutarate WT wild type Nth1 neutral trehalase pNth1 phosphorylated neutral trehalase DMSO dimethyl sulfoxide TCEP tris (2-carboxyethyl)phosphine VDM validoxylamine CD circular dichroism |
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