The C-terminal tail of Arabidopsis 14-3-3ω functions as an autoinhibitor and may contain a tenth α-helix

The eukaryotic regulatory protein 14-3-3 is involved in many important plant cellular processes including regulation of nitrate assimilation through inhibition of phosphorylated nitrate reductase (pNR) in darkened leaves. Divalent metal cations (Me²⁺) and some polyamines interact with the loop 8 region of the 14-3-3 proteins and allow them to bind and inhibit pNR in vitro. The role of the highly variant C-terminal regions of the 14-3-3 isoforms in regulation by polycations is not clear. In this study, we carried out structural analyses on the C-terminal tail of the Arabidopsis 14-3-3ω isoform and evaluated its contributions to the inhibition of pNR. Nested C-terminal truncations of the recombinant 14-3-3ω protein revealed that the removal of the C-terminal tail renders the protein partially Mg²⁺-independent in both pNR binding and inhibition of activity, suggesting that the C-terminus functions as an autoinhibitor. The C-terminus of 14-3-3ω appears to undergo a conformational change in the presence of polycations as demonstrated by its increased trypsin cleavage at Lys-247. C-terminal truncation of 14-3-3ω at Thr-255 increased its interaction with antibodies to the C-terminus of 14-3-3ω in non-denaturing conditions, but not in denaturing conditions, suggesting that the C-terminal tail contains ordered structures that might be disrupted by the truncation. Circular dichroism (CD) analysis of a C-terminal peptide, from Trp-234 to Lys-249, revealed that the C-terminal tail might contain a tenth α-helix, in agreement with the in silico predictions. The function of the putative tenth α-helix is not clear because substituting two prolyl residues within the predicted helix (E245P/1246P mutant), which prevented the corresponding peptide from adopting a helical conformation, did not affect the inhibition of pNR activity in the presence or absence of Mg²⁺. We propose that in the absence of polycations, access of target proteins to their binding groove in the 14-3-3 protein is restricted by the C-terminus, which acts as part of a gate that opens with the binding of polycations to loop 8.