STRUCTURAL STUDIES OF THE 14-3-3 PROTEIN AND NEUTRAL TREHALASE (Nth1) COMPLEX

Miroslava Alblova1, Aneta Smidova1, Petr Man2, Tomas Obsil3, Veronika Obsilova1

 

1Department of Structural Biology of Signaling Proteins, Division Biotechnology and Biomedicine Center of the Academy of Sciences and Charles University in Vestec (BIOCEV), Institute of Physiology, The Czech Academy of Sciences, Prumyslova 595, 25250 Vestec, Czech Republic

2Laboratory of Structural Biology and Cell Signaling, BIOCEV, Institute of Microbiology CAS, Prumyslova 595, 25250 Vestec, Czech Republic

3Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Hlavova 8, 12843 Prague, Czech Republic

 

 

            14-3-3 proteins are a family of highly conserved molecules which were found in all eukaryotes. They interact with and regulate the function of hundreds of different proteins by recognizing specific phosphoserine- or phosphothreonine-containing motifs. Thus 14-3-3 are involved in many important physiological processes such as: regulation of cell cycle, signal transduction, metabolism, gene transcription or apoptosis. We focused on understanding of the 14-3-3 protein function in the regulation of the neutral trehalase (Nth1, EC 3.2.1.28) from Saccharomyces cerevisiae. This enzyme hydrolyses disaccharide trehalose and helps yeasts to survive different stress conditions. Nth1 can be phosphorylated by PKA and/or CDK1. Its activity is enhanced by the yeast 14-3-3 protein (Bmh1) binding [1, 2] and/or by Ca2+ binding within the EF-hand-like motif containing domain [3].

            For revealing of the mechanism of the 14-3-3- and Ca-dependent activation of Nth1, solving the structure of Nth1 and its complex with 14-3-3 protein we used site-directed mutagenesis, enzyme activity measurements, MST, H/D exchange coupled to MS, SAXS and protein crystallography.

            Our crystal structure of full-length Nth1 in complex with 14-3-3 protein provides a detailed mechanistic insight into the role of 14-3-3 proteins in activating Nth1 [4]. We proved that 14-3-3 protein binding induces a rearrangement of the whole Nth1 molecule and enables the proper three-dimensional configuration of the pNth1 catalytic and calcium-binding domains relative to each other. The complex formation stabilizes the intrinsically disordered N-terminal part of Nth1 and moreover 14-3-3 protein directly interacts also with the separate Ca-binding domain of Nth1. Thus the EF-hand-like motif can function as an intermediary through which the 14-3-3 protein modulates the structure and function of the catalytic domain of Nth1. This process stabilizes the flexible part of Nth1 active site, so called “lid” loop, which completes the active site of pNth1 by providing side-chains important for catalysis and is crucial for the Nth1 activation. Our crystal structure of fully active Nth1 bound to yeast 14-3-3 protein Bmh1 provides the first high-resolution view of the neutral trehalase from eukaryotic organism as well as highlights the ability of 14-3-3 proteins to modulate the tertiary structure of a multi-domain binding partner.

 

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2. Macakova, E. Kopecka, M. Kukacka, Z. Veisova, D. Novak, P. Man, P. Obsil, T. Obsilova, V. BBA. – Gen. Subjects, 2013; 1830, 4491 – 4499.

3. Kopecka, M. Kosek, D. Kukacka, Z. Rezabkova, L. Man, P. Novak, P. Obsil, T. Obsilova, V.,  J. Biol. Chem., 2014, 289, 13948 – 13961.

4. Alblova, M. Smidova, A. Docekal, V. Vesely, J. Herman, P. Obsilova, V. Obsil, T., Proc. Natl. Acad. Sci. U. S. A., 2017, 114, E9811 – E9820.