Abstract
The leucine zipper (LZ) domain of the HY5 transcription factor from Arabidopsis thaliana has unique primary structural properties, including major occupation by the Leu residues as well as two buried polar residues in the a positions and a localized distribution of charged and polar residues in the first three heptad repeats. In this study, we solved the crystal structure of the HY5 LZ domain and show that the peculiarities in the primary sequence yield unusual structural characteristics. For example, the HY5 LZ domain exhibits a bipartite charge distribution characterized by a highly negative electrostatic surface potential in its N-terminal half and a nearly neutral potential in its C-terminal half. The LZ N-terminal region also contains two consecutive putative trigger sites for dimerization of the coiled coils. In addition, two buried asparagines at a positions 19 and 33 in the HY5 LZ domain display distinct modes of polar interaction. Whereas Asn(19) shows a conformational flip-flop, Asn(33) is engaged in a permanent hydrogen bond network. CD spectropolarimetry and analytical ultracentrifugation experiments performed with versions of the HY5 LZ domain containing mutations in the a positions yielded further evidence that position a amino acid residues are crucial for achieving an oligomeric state and maintaining stability. However, a low correlation between position a amino acid preference, core packing geometry, and rotamer conformations suggests that the oligomeric state of the LZ domain is not governed entirely by known structural properties. Taken together, our results suggest structural factors conferring conformational integrity of the HY5 LZ homodimer that are more complicated than proposed previously.
Highlights
Among coiled-coil proteins, the basic leucine zipper3 proteins play crucial roles in the regulation of transcription
The oligomeric state of HM1 and HM3 is dimeric, as is the case for the WT peptide (Table 4). These results indicate that HY5 shows only a weak preference of amino acid type in the a positions, which is consistent with the findings discussed above, and that the amino acid composition of position a in the HY5 leucine zipper (LZ) domain is optimized in terms of thermal stability of the dimer
The thermal profiles of LZ domains formed from Asn33 mutant peptides (HM5 and HM8) were shifted rightward and were more stable than those formed with Asn19 mutant peptides (HM4 and HM7). These findings indicate that the HY5 LZ domain is more stabilized when Asn33 is replaced by hydrophobic residues than when Asn19 is substituted with hydrophobic residues
Summary
Peptide Synthesis—N-terminally acetylated synthetic peptides were purchased from Peptron, Inc. (Daejeon, South Korea). The resulting cell lysates were subjected to centrifugation at 15,000 rpm for 1 h; the clarified supernatants were filtered using 0.45-m filters (Sartorius AG); and the HY5 LZ domain-GST fusion proteins were purified using a GST affinity column (Amersham Biosciences). The selenomethionine (SeMet)-containing WT HY5 LZ domain was prepared from E. coli strain BL21 grown in M9 medium [16] supplemented with 2 mM MgSO4, 0.4% (w/v) glucose, and 0.1 mM CaCl2 When this culture reached A600 ϳ 0.8 at 37 °C, 100 mg each of Leu, Ile, Val, Thr, Phe, and Lys and 50 mg of L-(ϩ)-SeMet (Sigma) were added per liter of growth medium. When the SeMet-containing version of the WT HY5 LZ domain was purified, 5 mM dithiothreitol was added to the buffers used for a native sample preparation. All non-glycine amino acid residues were located in the most favored region of the Ramachandran plot calculated with the refined model
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