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Protein Science, Vol 4, Issue 3 405-415, Copyright © 1995 by Cold Spring Harbor Laboratory Press

ARTICLE

Conformationally constrained analogs of protein kinase inhibitor(6-22)amide: Effect of turn structures in the center of the peptide on inhibition of cAMP-dependent protein kinase

D. B. GLASS, J. TREWHELLA, R. D. MITCHELL and D. A. WALSH
Departments of Pharmacology and Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322

The high-affinity interaction between protein kinase inhibitor (PKI)(6-22)amide (Thr(6)-Tyr-Ala-Asp-Phe-Ile-Ala- Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile(22)-NH(2)) and the catalytic subunit of cAMP-dependent protein kinase requires both the N-terminal Thr(6) to Ile(11) sequence of the inhibitor peptide and its C-terminal pseudosubstrate site comprised of Arg(15) to Ile(22). Small angle X-ray scattering data indicate that PKI(6-22)amide has a compact, rather than extended, structure in solution (Reed J et al., 1989, Biochem J 264:371-380). CD spectroscopic analysis of the PKI peptide led to the suggestion that a {beta}-turn structure might be located in the -Ala(12)-Ser-Gly-Arg(15)-connecting sequence in the middle of the molecule (Reed J, Kinzel V, Cheng HC, Walsh DA, 1987, Biochemistry 26:7641-7647). To investigate this possibility further, conformationally constrained and flexible analogs of PKI(6-22)amide were synthesized and used to study the structure-function relationships of this central portion of the inhibitor. (Des 12-14)PKI(6-22) amide exhibited over a 200-fold loss in inhibitory activity. Replacement of the omitted -Ala(12)-Ser-Gly(14)-sequence with aminocaprylic acid yielded an analog that regained more than 90% of the lost binding energy. The D-alanine(14) PKI analog was as potent as the parent peptide, whereas the {beta}-alanine(14) and the sarcosine(14) analogs were only 10-fold less active. Several peptides that promoted a {beta}-turn structure at residues 12-15 showed about 200-fold decreases in inhibitory activity. Two constrained analogs that could not assume a {beta}-turn conformation were only 30-fold less potent than PKI(6-22)amide. Thus, the structure of the central connecting portion of the PKI peptide, encompassing residues 12-15, greatly influences its ability to effectively bind to and inhibit the catalytic subunit. We conclude, however, that a formal {beta}-turn at this position is not required and is actually detrimental for a high-affinity interaction of PKI(6-22)amide with the enzyme. These results are interpreted in light of the Fourier-transform infrared spectra of the peptide analogs and the crystal structure of the peptide bound at the active site of the protein kinase (Knighton DR et al., 1991b, Science 253:414-420).
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