Structures of Protein Kinases

The first crystal structure of a protein kinase to be determined was that of the catalytic domain of cyclic-AMP-dependent kinase, also known as protein kinase A (PKA) [8,9]. PKA is a ubiquitously expressed Ser/Thr kinase involved in several different signaling pathways. The overall fold of the catalytic domain, first seen for PKA, is highly conserved among all Ser/Thr and tyrosine protein kinases. Additional sequences and domains that are located both C-terminal and N-terminal to this structurally conserved catalytic domain account for most of the functional diversity among different protein kinases.

The structure of the catalytic domain of PKA, co-crystallized with adenosine triphosphate (ATP)-Mg and an inhibitor peptide molecule, is shown in Fig. 1 [8,9]. The protein kinase domain is composed of two lobes with an overall length of roughly 275 residues. The N-terminal lobe, alternatively referred to as the N lobe or the small lobe, contains an anti-parallel P-sheet and one important a-helix (helix C), while the C-terminal lobe (or large lobe) is primarily a-helical in composition.

This structure is an example of a protein kinase that is in a catalytically competent conformation and serves as a model for the structure of active protein kinases. The two substrates of the phosphorylation reaction, ATP and the polypeptide phosphate acceptor, both bind in a cleft formed between the N lobe and the C lobe. The highly charged chemical groups of the ATP and two associated Mg2+ ions are coordinated by a collection of highly conserved Lys, Asn, Asp, and Phe residues (Fig. 2). A recent crystal structure of PKA with a transition state analog supports an in-line mechanism of phosphate transfer from ATP to a target Ser-containing substrate [10]. This mechanism is believed to be conserved among protein kinases, consistent with the highly conserved kinase fold.

Figure 1 Structure of the Ser/Thr kinase PKA complexed with ATP-Mg2+ and a specific inhibitor peptide (PDB code 1ATP) [8,9]. ATP is gray, the peptide inhibitor PKI is yellow, a-helix C is pink, and the activation loop is red.

The structure of the catalytic domain of Lck [11] provides an example of the catalytically active conformation of a tyrosine kinase enzyme. Despite the absence of both nucleotide and substrate, the conformations of key catalytic residues are primed for catalysis, in line with the conformations of homologous residues seen in the structure of PKA. The active form of the kinase domain of Lck is phosphory-lated on Tyr 394, which lies in the activation loop of the kinase domain, a segment of roughly 15 to 20 residues located between the N and C lobes of the kinase domain. Phosphorylation on one or more residues in the activation loop is a signature of the activated state of many protein kinases. In active Lck, phosphorylated Tyr 394 forms a salt bridge with Arg 387, thereby stabilizing the conformation of the activation loop and locking the enzyme in a catalytically active state.

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