Mapping of the interaction surface of phosducin using HDX-MS


Miroslava Kacirova1,2, Petr Man1,3, Alan Kadek3 and Tomas Obsil1,2


1Faculty of Science, Charles University in Prague, 12843 Prague, Czech Republic
2Institute of Physiology, Academy of Sciences of Czech Republic, 14220 Prague, Czech Rep.
3Institute of Microbiology, Academy of Sciences of Czech Republic, 14220 Prague, Czech Rep.
mirkakacirova@seznam.cz

 

Phosducin (Pdc), a regulatory and highly conserved phosphoprotein, plays an important role in the regulation of the G protein signaling by modulating an amount of the Gtabg heterotrimer through a competition with the Gta subunit for binding to the Gtbg complex [1]. Besides its well-established role in the regulation of the G protein signaling, Pdc is also involved in the transcriptional control and the modulation of blood pressure. The function of Pdc is regulated through its phosphorylation and a binding to the regulatory 14-3-3 protein [2]. The 14-3-3 proteins are scaffolding molecules that regulate the function of other proteins through a number of different mechanisms. The exact role of the 14-3-3 protein in the regulation of Pdc function is, however, still elusive.

We have recently shown that the 14-3-3z protein interacts with and reduces the flexibility of both the N-terminal and the C-terminal domains of doubly phosphorylated Pdc (dpPdc). We also observed that the phosphorylation alone has a significant effect on the structure of the whole Pdc molecule [3]. In order to gain additional insight into the interaction of Pdc with the 14-3-3 protein, we decided to use hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) to map their interaction interface. Therefore, the HDX kinetics of the 14-3-3:dpPdc and 14-3-3:Pdc complexes as well as free 14-3-3, dpPdc and Pdc proteins were measured. The exchange kinetics of Pdc regions were followed on 72 peptides from the pepsin digest, together covering 100 % of its sequence, while the exchange kinetics of 14-3-3 regions were followed on 94 peptides covering 100 % of its sequence. Our data revealed that several dpPdc and 14-3-3 peptides exhibit significant change in the deuteration kinetics upon the complex formation suggesting that these regions either form the binding interface or undergo a structural change upon the interaction. The slower deuterium incorporation was observed for 14-3-3 peptides 48-59, 60-66, 155-174, 191-199. These peptides map to the surface of helix H3 as well as outside the central channel of the 14-3-3 protein dimer. In the case of dpPdc the slower deuterium incorporation was observed for peptides 4-28, 117-135, 152-157, 208-212, 221-242. These peptides map to the surface of both domains. Interestingly, HDX-MS data also show significant differences between dpPdc and Pdc confirming that phosphorylation alone has significant effect on Pdc structure.

This work was funded by Grant P305/11/0708 of the Czech Science Foundation, by Research Project MSM0021620857 and by Research Project AV0Z50110509 of the Academy of Sciences of the Czech Republic.

 

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