Solution Structure of Porcine Delta Sleep-inducing Peptide Immunoreactive Peptide A Homolog of the ShortsightedGene Product

Gabi Seidel,Knut Adermann,Thomas Schindler,Andrzej Ejchart,Rainer Jaenicke,Wolf-Georg Forssmann,Paul Rösch

doi:10.1074/jbc.272.49.30918

Gabi Seidel, Knut Adermann + Show 5 more

Open Access

https://doi.org/10.1074/jbc.272.49.30918

Copy DOI

Abstract

The 77-residue delta sleep-inducing peptide immunoreactive peptide (DIP) is a close homolog of the Drosophila melanogaster shortsighted gene product. Porcine DIP (pDIP) and a peptide containing a leucine zipper-related partial sequence of pDIP, pDIP(9-46), was synthesized and studied by circular dichroism and nuclear magnetic resonance spectroscopy in combination with molecular dynamics calculations. Ultracentrifugation, size exclusion chromatography, and model calculations indicated that pDIP forms a dimer. This was confirmed by the observation of concentration-dependent thermal folding-unfolding transitions. From CD spectroscopy and thermal folding-unfolding transitions of pDIP(9-46), it was concluded that the dimerization of pDIP is a result of interaction between helical structures localized in the leucine zipper motif. The three-dimensional structure of the protein was determined with a modified simulated annealing protocol using experimental data derived from nuclear magnetic resonance spectra and a modeling approach based on an established strategy for coiled coil structures. The left-handed super helical structure of the leucine zipper type sequence resulting from the modeling approach is in agreement with known leucine zipper structures. In addition to the hydrophobic interactions between the amino acids at the heptade positions a and d, the structure of pDIP is stabilized by the formation of interhelical i to i' + 5 salt bridges. This result was confirmed by the pH dependence of the thermal-folding transitions. In addition to the amphipatic helix of the leucine zipper, a second helix is formed in the NH2-terminal part of pDIP. This helix exhibits more 310-helix character and is less stable than the leucine zipper helix. For the COOH-terminal region of pDIP no elements of regular secondary structure were observed.

Highlights

PDIP shares significant homology with the murine TSC-22 protein and the product of the Drosophila melanogaster shortsighted gene, and the regions upstream of the leucine zipper are almost identical
TSC-22 was reported to be present in both the cytoplasmic and the nuclear fraction, and it has been discussed to function as a transcriptional regulator that is activated by transcription growth factor-␤1 and other growth factors of osteoblastic cells [6]
The resin was washed with trifluoroacetic acid, and the crude peptide was precipitated by addition of chilled tert-butylmethyl ether, washed with tert-butylmethyl ether, and lyophilized from 5% acetic acid (crude yields: Porcine DIP (pDIP), 516 mg, 39.7%; pDIP(9 – 46), 225 mg, 38.7%)

Summary

MATERIALS AND METHODS

Solid-phase Peptide Synthesis—Peptides were assembled using Fmoc chemistry on an automated peptide synthesizer (model 9050, PerSeptive Biosystems, Wiesbaden, Germany). Fmoc amino acids were purchased from Orpegen (Heidelberg, Germany) and PerSeptive Biosystems. Acylations were performed in 30 min with a 4-fold excess of Fmoc-L-amino acid in the presence of TBTU/diisopropylethylamine/ 1-hydroxybenzotriazole (4 eq). After deprotection of the terminal amino groups, the peptidyl resins were acetylated with a mixture of dichloromethane/N,N-dimethylformamide/ acetic anhydride/pyridine (40:40:19:1, volume) in 20 min at 0 °C. Wavelength scans were performed at discrete temperatures from 25 to 90 °C in a thermostatically controlled quartz cell of 0.5 or 0.05 cm pathlength, depending on peptide concentration.

Solution Structure of pDIP

RESULTS AND DISCUSSION

RMSD among backbone structures