Monitoring of phosphorylation progress of tyrosine hydroxylase and interaction with 14‑3‑3z elucidated by NMR

Petr Louša, Hana Nedozrálová, Jiří Nováček, Lukáš Hason, Jozef Hritz
hritz
@ceitec.muni.cz

Department of Structural Biology, CEITEC MU, Kamenice 753/5, 625 00 Brno, Czech Republic

 

Human tyrosine hydroxylase 1 (hTH1) activity is regulated by phosphorylation of its N-terminus and by an interaction with the modulator 14‑3‑3 proteins. In order to monitor the structural changes within the regulatory domain of hTH1 (RD-hTH1, region of first 169 residues) caused by phosphorylation of S19 and S40 we have assigned NMR spectra by two different approaches. The non-uniform sampling approach (NUS) based on sparse multidimensional Fourier transform allows efficient acquisition of high dimensional NMR spectra. Increased dimensionality (5D) provided significant speed up of backbone and side-chain assignment of the unstructured RD-hTH1 region (about first 70 residues). The rest (structured parts) of RD-hTH1 was assigned by conventional set of 3D NMR experiments.

The quantification of quite complex binding mechanism between the phosphorylated hTH1 and 14-3-3zeta required novel approach. We used 31P NMR to investigate interactions between 14-3-3z and minimalistic system comprising first 50 residues of hTH1 (hTH1_50), containing both phosphorylation sites of our interest (S19, S40). Dissociation constants between 14-3-3z and singly phosphorylated hTH1_50 (pS19 or pS40) were determined. Analysis of the NMR titration data revealed that a 14-3-3z dimer and the S19_S40-doubly phosphorylated hTH1_50 interact in multiple ways, with three major complexes formed. As the proof of principle of this methodology was successfully shown for hTH1_50 we continue in applying it for revealing of binding scenario between 14-3-3z  and phosphorylated regulatory domain of hTH1 protein.

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The project is financed from the SoMoPro II programme. The research leading to this invention has acquired a financial grant from the People Programme (Marie Curie action) of the Seventh Framework Programme of EU according to the REA Grant Agreement No. 291782. The research is further co-financed by the South-Moravian Region. The article/paper reflects only the author's views and the Union is not liable for any use that may be made of the information contained therein. In addition, this work was also supported by Czech Science Foundation (I 1999-N28). This work was realized in CEITEC – Central European Institute of Technology with research infrastructure supported by the project CZ.1.05/1.1.0.0/02.0068 financed from European Regional Development Fund.