Poly-l-proline type II peptide mimics as probes of the active site occupancy requirements of cGMP-dependent protein kinase

: Based on the X‐ray crystal structure of cAMP‐dependent protein kinase (PKA) with the endogenous inhibitor PKI and the X‐ray crystal structure of cyclin‐dependent kinase 2 (CDK2) with a substrate peptide, a proposal is put forth that some protein kinases bind peptide substrates in their active sites in the poly‐l‐proline type II (PPII) conformation. In this work, PPII peptide mimics are evaluated as pseudosubstrate inhibitors of cGMP‐dependent protein kinase (PKG) to explore if PKG also binds peptide substrates in the PPII conformation. Inhibition data of our PPII mimetics provide evidence that the P − 1, P − 2, and P − 3 residues of substrate peptides bind in the PPII conformation (φ approximately −75°, ψ approximately 145°). In addition, the inhibition data also suggest that the P − 1, P − 2, and P − 3 residues in substrate peptides bind with a gauche(−) χ1 angle.