14-3-3
PROTEIN C-TERMINAL STRETCH OCCUPIES LIGAND BINDING GROOVE AND IS DISPLACED BY
PHOSPHOPEPTIDE BINDING
J. Šilhán1,4, V. Obšilová4, J. Večeř3,
P. Heřman3, M. Šulc2,5, J. Teisinger4, and T.
Obšil1,4*
1Department of Physical and Macromolecular Chemistry, Faculty of Science,
Charles University, 128 43 Prague,
2Department of Biochemistry, Faculty of Science, Charles University, 128
43 Prague,
3Institute of Physics, Faculty of Mathematics and Physics, Charles
University, 121 16 Prague,
4Institute of Physiology, Academy of Sciences of the Czech Republic, 142
20 Prague,
5Institute of Microbiology, Academy of Sciences of the Czech Republic, 142
20 Prague, Czech Republic
14-3-3
proteins are important regulators of numerous cellular signaling circuits. They
bind to phosphorylated protein ligands and regulate their functions by a number
of different mechanisms [1,2]. The C-terminal part of 14-3-3 protein is known
to be involved in the regulation of 14-3-3 binding properties. The structure of
this region is unknown; however possible location of the C-terminal stretch
within the ligand binding groove of 14-3-3 protein has been suggested [3,4]. In order to fully understand the role of the C-terminal stretch in
the regulation of 14-3-3 protein binding properties we investigated the
physical location of the C-terminal stretch and its changes upon the ligand
binding. For this purpose we have used Förster resonance energy transfer (FRET)
measurements and molecular dynamics simulation. FRET measurements between Trp242
located at the end of the C-terminal stretch and a dansyl group attached at two
different cysteine residues (Cys25 or Cys189) indicate
that in the absence of the ligand the C-terminal stretch occupies the ligand
binding groove of 14-3-3 protein [5]. Our data also show that phosphopeptide
binding displaces the C-terminal stretch from the ligand binding groove.
Intramolecular distances calculated from FRET measurements fit well with
distances obtained from molecular dynamics simulation of full length 14-3-3z protein.
This work
was supported by Grants 204/03/0714 and 309/02/1479 of the Grant Agency of the
Czech Republic; by Grant B5011308 of the Grant Agency of the Czech Academy of
Sciences; by Research Projects 1K03020, MSM 1131 00001, and 1132 00001 of the
Ministry of Education, Youth and Sports of the Czech Republic, and by Research
Project AVOZ 5011922.
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3.
D.
Liu, J. Bienkowska, C. Petosa, R. J. Collier, H. Fu & R. Liddington, Nature, 376 (1995) 191-194.
4. V. Obsilova, P. Herman, J.Vecer, M. Sulc, J. Teisinger & T. Obsil, J. Biol. Chem., 279 (2004) 4531-4540.
5. J. Silhan, V. Obsilova, J. Vecer, P. Herman, M. Sulc, J. Teisinger & T. Obsil, J. Biol. Chem., 279 (2004) 49113-49119.