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The C‐terminal tail of Arabidopsis 14‐3‐3ω functions as an autoinhibitor and may contain a tenth α‐helix
Author(s) -
Shen Wei,
Clark A. Clay,
Huber Steven C.
Publication year - 2003
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.2003.01739.x
Subject(s) - c terminus , arabidopsis , gene isoform , circular dichroism , biochemistry , n terminus , peptide , cleavage (geology) , biophysics , chemistry , biology , peptide sequence , stereochemistry , amino acid , mutant , gene , paleontology , fracture (geology)
Summary 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 (Me 2+ ) 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 Mg 2+ ‐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/I246P 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 Mg 2+ . 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.