Structural basis of the 14-3-3 protein-dependent activation of yeast neutral trehalase Nth1

Abstract

Background: Trehalases 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.

Methods: To 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.

Results: Our 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.

Conclusions: The 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 significance: The 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.

Keywords: 14-3-3; Bmh; CD; Circular dichroism; DMSO; DSG; DSS; H/D exchange; H/D exchange coupled to mass spectrometry; HDX; HDX–MS; Molecular modeling; Neutral trehalase; Nth1; TCEP; VDM; WT; circular dichroism; dimethyl sulfoxide; disuccinimidyl glutarate; disuccinimidyl suberate; neutral trehalase; pNth1; phosphorylated neutral trehalase; tris (2-carboxyethyl)phosphine; validoxylamine; wild type.