Isoleucine 368 is involved in low-affinity binding of N6-modified cAMP analogues to site B of the regulatory subunit of cAMP-dependent protein kinase I.
The regulatory (R) subunit of cAMP-dependent protein kinase has a well-defined domain structure including the two in-tandem cAMP-binding sites that constitute the C-terminus of the protein. The N-terminal binding site (A) has a considerably higher affinity for analogues of cAMP that are substituted with bulky and hydrophobic substituents at the 6-amino group of the adenine ring compared to the affinity observed at the second site (B). On the basis of the crystal structure of the catabolite gene activator protein from Escherichia coli, molecular modelling of the binding domains suggested that a tyrosine (Y244) in site A could be involved in a high-affinity hydrophobic interaction, whereas a corresponding isoleucine (I368) in domain B could lead to steric hindrance in the binding of bulky N6-substituted analogues. Site-directed mutagenesis was used to construct mutations in Y244 and I368. Binding displacement experiments showed that replacing the tyrosine in site A with isoleucine (Y244I) did not affect the interaction of either N6-substituted or otherwise modified analogues with this site. However, replacing I368 with tyrosine (I368Y) led to a 3-4-fold increase in affinity for those N6-modified analogues that had a hydrophobic group attached directly or close to the 6-amino molecule. We conclude that I368 is involved in the molecular interaction between binding domain B and the 6-amino group of the adenine moiety of cAMP and that this residue is partly responsible for the reduced affinity of N6-substituted cAMP analogues for this site.
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University of Bergen, Center of Molecular Medicine, Haukeland Hospital, Norway.
Dostmann
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WR
Ogreid
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D018076:DNA, Complementary; D004926:Escherichia coli; D007532:Isoleucine; D046911:Macromolecular Substances; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D018076:DNA, Complementary; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D007532:Isoleucine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases