Crystal structures of the myristylated catalytic subunit of cAMP‐dependent protein kinase reveal open and closed conformations

Abstract

Three crystal structures, representing two distinct conformational states, of the mammalian catalytic subunit of CAMP-dependent protein kinase were solved using molecular replacement methods starting from the refined structure of the recombinant catalytic subunit ternary complex (Zheng, J., et al., 1993a, Biochemistry 32,2154-2161). These structures correspond to the free apoenzyme, a binary complex with an iodinated inhibitor peptide, and a ternary complex with both ATP and the unmodified inhibitor peptide. The apoenzyme and the binary complex crystallized in an open conformation, whereas the ternary complex crystallized in a closed conformation similar to the ternary complex of the recombinant enzyme. The model of the binary complex, refined at 2.9 A 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-terminal30 residues. This rotation of the small lobe alters the cleft interface in the active-site region surrounding the glycine-rich loop and Thr 197, a critical phosphorylation site. 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 by four segments of the protein that are widely dispersed in the linear sequence. The N-terminal40 residues that lie outside the conserved catalytic core are anchored by the N-terminal myristylate plus an amphipathic helix that spans both lobes and is capped by Trp 30. Both posttranslational modifications, phosphorylation and myristylation, contribute directly to the stable structure of this enzyme.

Unveiling the Substrate Specificity of Meprin β on the Basis of the Site in Protein Kinase A Cleaved by the Kinase Splitting Membranal Proteinase

“…(d) KSMP was found to cleave the Csubunit in its native conformation but not if the kinase is pre-denatured (2). (e) It distinguishes between the "open" and "closed" conformations of the C-subunit (4) that were recently identified by x-ray crystallography of this kinase (5).…”

Molecular Heterogeneity of Phospholipase D (PLD)

“…Myristoylation of many proteins is thought to represent a mechanism for reversible membrane association (26 -28). It is believed that the hydrophobic nature of myristate allows it to associate directly with membrane lipids, but there are proteins, such as mammalian cAMP-dependent kinase, in which myristoylation has no membrane-targeting function, but is instead important for structural stabilization (29). In some cases, it has been reported that myristate interacts with a specific membrane protein, thus mediating a targeted protein-protein interaction (30).…”

Proteus in the World of Proteins: Conformational Changes in Protein Kinases

“…It is a close approximation of the conformation adopted by PKA at the moment the phosphotransfer is taking place and is merely identical to the PKA-ATP complex structure. The same group showed that the N-and Clobes of PKA can rotate relative to each other around Gly125 in the hinge region [12], a short strand connecting the two lobes opening the catalytic cleft. This movement displaces the catalytic amino acids from the enzymatically competent conformation they adopt in the closed structure described above.…”

“…NMR experiments in the presence and absence of myristate suggest the existence of a similar binding site in phosphorylated Src [64]. Several other kinases such as p38a [68] and PKA [12] have also been shown to bind lipids, but the biological meaning of this event is not yet clear. Mutations affecting SH domain binding can cause diseases by disrupting kinase regulation [69].…”