CRYSTAL-STRUCTURES OF THE MYRISTYLATED CATALYTIC SUBUNIT OF CAMP-DEPENDENT PROTEIN-KINASE REVEAL OPEN AND CLOSED CONFORMATIONS

Three crystal structures, representing two distinct conformational sta tes, of the mammalian catalytic subunit of cAMP-dependent protein kina se were solved using molecular replacement methods starting from the r efined structure of the recombinant catalytic subunit ternary complex (Zheng, J., et al., 1993a, Biochemistry 32, 2154-2161). These structur es correspond to the free apoenzyme, a binary complex with an iodinate d inhibitor peptide, and a ternary complex with both ATP and the unmod ified inhibitor peptide. The apoenzyme and the binary complex crystall ized in an open conformation, whereas the ternary complex crystallized in a closed conformation similar to the ternary complex of the recomb inant enzyme. The model of the binary complex, refined at 2.9 angstrom resolution, shows the conformational changes associated with the open conformation. These can be described by a rotation of the small lobe and a displacement of the C-terminal 30 residues. This rotation of the small lobe alters the cleft interface in the active-site region surro unding the glycine-rich loop and Thr 197, a critical phosphorylation s ite. In addition to the conformational changes, the myristylation site , absent in the recombinant enzyme, was clearly defined in the binary complex. The myristic acid binds in a deep hydrophobic pocket formed b y four segments of the protein that are widely dispersed in the linear sequence. The N-terminal 40 residues that lie outside the conserved c atalytic core are anchored by the N-terminal myristylate plus an amphi pathic helix that spans both lobes and is capped by Trp 30. Both postt ranslational modifications, phosphorylation and myristylation, contrib ute directly to the stable structure of this enzyme.