Properties of the monomeric form of human 14-3-3zeta protein and its interaction with tau and HspB6.

Sluchanko NN; Sudnitsyna MV; Seit-Nebi AS; Antson AA; Gusev NB

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Article

Title:	Properties of the monomeric form of human 14-3-3zeta protein and its interaction with tau and HspB6.
Authors:	Sluchanko NN; Sudnitsyna MV; Seit-Nebi AS; Antson AA; Gusev NB
Publication:	Biochemistry. 2011 Nov 15;50(45):9797-808. doi: 10.1021/bi201374s. Epub 2011 Oct 19.
PubmedID	21978388
Abstract
Dimers formed by seven isoforms of the human 14-3-3 protein participate in multiple cellular processes. The dimeric form has been extensively characterized; however, little is known about the structure and properties of the monomeric form of 14-3-3. The monomeric form is involved in the assembly of homo- and heterodimers, which could partially dissociate back into monomers in response to phosphorylation at Ser58. To obtain monomeric forms of human 14-3-3zeta, we produced four protein constructs with different combinations of mutated (M) or wild-type (W) segments E(5), (12)LAE(14), and (82)YREKIE(87). Under a wide range of expression conditions in Escherichia coli, the MMM and WMM mutants were insoluble, whereas WMW and MMW mutants were soluble, highly expressed, and purified to homogeneity. WMW and MMW mutants remained monomeric over a wide range of concentrations while retaining the alpha-helical structure characteristic of wild-type 14-3-3. However, WMW and MMW mutants were highly susceptible to proteolysis and had much lower thermal stabilities than the wild-type protein. Using WMW and MMW mutants, we show that the monomeric form interacts with the tau protein and with the HspB6 protein, in both cases forming complexes with a 1:1 stoichiometry, in contrast to the 2:1 and/or 2:2 complexes formed by wild-type 14-3-3. Significantly, this interaction requires phosphorylation of tau protein and HspB6. Because of minimal changes in structure, MMW and especially WMW mutant proteins are promising candidates for analyzing the effect of monomerization on the physiologically important properties of 14-3-3zeta.