Solution structure of synthetic peptide inhibitor and substrate of cAMP-dependent protein kinase. A study by 2D 1H NMR and molecular dynamics |
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Authors: | ANDR PADILLA JENNIFER A HAUER IGOR TSIGELNY JOSEPH PARELLO SUSAN S TAYLOR |
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Institution: | ANDRÉ PADILLA,JENNIFER A. HAUER,IGOR TSIGELNY,JOSEPH PARELLO,SUSAN S. TAYLOR |
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Abstract: | Peptides derived from the inhibitor of cAMP-dependent protein kinase, PKI, have been studied by 2D 1H NMR techniques. These include the inhibitor PKI(6-22), the substrate Ala20-Ser21] PKI(5-24), and a phosphorylated form of the latter Ala20-Ser21P] PKI(5-24). A homologous fold was found in the three peptides which consisted of an N-terminal segment in helical conformation to residue 13 and a C-terminal segment poorly defined conformationally. A parallel study was carried out by molecular dynamics (MD) for the inhibitor peptide PKI (5-24). The N-terminal helix, as observed in the crystal structure of the catalytic subunit-PKI(5-24) complex, was conserved in the MD simulations with the enzyme-free inhibitor. Similarly the Gly14-Gly17 turn was apparent 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 between Asp9 and Arg15. Indeed, a detailed analysis of the NOE contacts by NOESY at low temperature (2°C) shows the occurrence of pH-dependent contacts with Phe10. We conclude that the binding of short inhibitors, such as PKI (5-24), to the enzyme involves a conformational rearrangement of the C-terminal region. The substrate Ala20-Ser21] PKI (5-24) and the product Ala20-Ser21P] PKI(5-24), give very similar structures with local rearrangements involving some of the side chains. © Munksgaard 1997. |
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Keywords: | cAMP-dependent protein kinase PKIz NMR molecular dynamics |
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