SOLUTION STRUCTURE OF SYNTHETIC PEPTIDE INHIBITOR AND SUBSTRATE OF CAMP-DEPENDENT PROTEIN-KINASE - A STUDY BY 2D H-1-NMR AND MOLECULAR-DYNAMICS

Peptides derived from the inhibitor of cAMP-dependent protein kinase, PKI, have been studied by 2D H-1 NMR techniques. These include the inh ibitor PKI(6-22), the substrate [Ala(20)-Ser(21)]PKI(5-24), and a phos phorylated form of the latter [Ala(20)-Ser(21)P]PKI(5-24). A homologou s fold was found in the three peptides which consisted of an N-termina l segment in helical conformation to residue 13 and a C-terminal segme nt poorly defined conformationally. A parallel study was carried out b y molecular dynamics (MD) for the inhibitor peptide PKI(5-24). The N-t erminal helix, as observed in the crystal structure of the catalytic s ubunit-PKI(5-24) complex, was conserved in the MD simulations with the enzyme-free inhibitor. Similarly the Gly(14)-Gly(17) turn was apparen t in all MD structures, whereas the C-terminal region, residues 18-24, was directed towards the N-terminal helix in contrast to the extended conformation of this segment pointing away from the N-terminal helix in the crystal structure. This is primarily due to ionic interaction b etween Asp(9) and Arg(15). Indeed, a detailed analysis of the NOE cont acts by NOESY at low temperature (2 degrees C) shows the occurrence of pH-dependent contacts with Phe(10). We conclude that the binding of s hort inhibitors, such as PKI(5-24), to the enzyme involves a conformat ional rearrangement of the C-terminal region. The substrate [Ala(20)-S er(21)]PKI(5-24) and the product [Ala(20)-Ser(21)P]PKI(5-24), give ver y similar structures with local rearrangements involving some of the s ide chains. (C) Munksgaard 1997.