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Sci Rep. 2019 Oct 18;9(1):15007. doi: 10.1038/s41598-019-50941-3.

Concatenation of 14-3-3 with partner phosphoproteins as a tool to study their interaction.

Author information

1
A.N. Bach Institute of Biochemistry, Federal Research Center of Biotechnology of the Russian Academy of Sciences, 119071, Moscow, Russia.
2
Department of Biochemistry, School of Biology, M.V. Lomonosov Moscow State University, 119991, Moscow, Russia.
3
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR, 97331, USA.
4
Laboratory for Biocrystallography, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, 3000, Leuven, Belgium.
5
A.N. Bach Institute of Biochemistry, Federal Research Center of Biotechnology of the Russian Academy of Sciences, 119071, Moscow, Russia. nikolai.sluchanko@mail.ru.
6
Department of Biophysics, School of Biology, M.V. Lomonosov Moscow State University, 119992, Moscow, Russia. nikolai.sluchanko@mail.ru.

Abstract

Regulatory 14-3-3 proteins interact with a plethora of phosphorylated partner proteins, however 14-3-3 complexes feature intrinsically disordered regions and often a transient type of interactions making structural studies difficult. Here we engineer and examine a chimera of human 14-3-3 tethered to a nearly complete partner HSPB6 which is phosphorylated by protein kinase A (PKA). HSPB6 includes a long disordered N-terminal domain (NTD), a phosphorylation motif around Ser16, and a core α-crystallin domain (ACD) responsible for dimerisation. The chosen design enables an unstrained binding of pSer16 in each 1433 subunit and secures the correct 2:2 stoichiometry. Differential scanning calorimetry, limited proteolysis and small-angle X-ray scattering (SAXS) support the proper folding of both the 14-3-3 and ACD dimers within the chimera, and indicate that the chimera retains the overall architecture of the native complex of 14-3-3 and phosphorylated HSPB6 that has recently been resolved using crystallography. At the same time, the SAXS data highlight the weakness of the secondary interface between the ACD dimer and the C-terminal lobe of 14-3-3 observed in the crystal structure. Applied to other 14-3-3 complexes, the chimeric approach may help probe the stability and specificity of secondary interfaces for targeting them with small molecules in the future.

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