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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
Author(s) -
Glass David B.,
Trewhella Jill,
Mitchell Ryan D.,
Walsh Donal A.
Publication year - 1995
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560040307
Subject(s) - peptide , chemistry , stereochemistry , alanine , amide , protein kinase a , peptide sequence , kinase , biochemistry , amino acid , gene
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 β‐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. (Des12–14)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 β‐alanine 14 and the sarcosine 14 analogs were only 10‐fold less active. Several peptides that promoted a β‐turn structure at residues 12–15 showed about 200‐fold decreases in inhibitory activity. Two constrained analogs that could not assume a β‐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 β‐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 255 :414–420).