Properties of the Monomeric
Form of Human 14-3-3ζ Protein and Its Interaction with Tau and
HspB6
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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 14-3-3ζ, we produced
four protein constructs with different combinations of mutated (M)
or wild-type (W) segments E<sup>5</sup>, <sup>12</sup>LAE<sup>14</sup>, and <sup>82</sup>YREKIE<sup>87</sup>. Under a wide range of expression
conditions in <i>Escherichia coli</i>, 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
α-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 14-3-3ζ