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.
The high-affinity interaction between protein kinase inhibitor (PKI)(6-22)amide(Thr6-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly- Arg-Arg-Asn- Ala-Ile22-NH2) and the catalytic subunit of cAMP-dependent protein kinase requires both the N-terminal Thr6 to Ile11 sequence of the inhibitor peptide and its C-terminal pseudosubstrate site comprised of Arg15 to Ile22. 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 -Ala12-Ser-Gly-Arg15-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 -Ala12-Ser-Gly14-sequence with aminocaprylic acid yielded an analog that regained more than 90% of the lost binding energy. The D-alanine14 PKI analog was as potent as the parent peptide, whereas the beta-alanine14 and the sarcosine14 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).
Glass
D B
DB
Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Trewhella
J
J
Mitchell
R D
RD
Walsh
D A
DA
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D047908:Intracellular Signaling Peptides and Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012996:Solutions; D017550:Spectroscopy, Fourier Transform Infrared; D013329:Structure-Activity Relationship; D014867:Water / Q000737:chemistry
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D012996:Solutions; C094363:protein kinase inhibitor (6-22)-amide; D014867:Water; D017868:Cyclic AMP-Dependent Protein Kinases