Kinases crystallization

Figure 2 Depiction of the active ( open ) and inactive ( closed ) conformations of Src kinase based on the analysis of x-ray structures of c-Src tyrosine kinase crystallized in its inactive state [7]. The stabilization of the inactive conformation is influenced by multiple events including intramolecular binding of the tyrosine-phosphorylated C-terminus tail to the SH2 domain as well as interactions between the SH3 domain and the SH2-kinase linker. CT, C-terminal NT, N-terminal. 

Fig. 14.1. Ribbon structure (magenta) of the phosphorylase kinase crystal structure 2PHK (20) bound with ATP (green carbons, colored by atom type) and substrate peptide (light blue ribbon). The N- and C-terminal lobes are highlighted the hinge region is shown in cyan, the a-C helix in gray, and the -loop in orange.

One of the most challenging aspects of obtaining a kinase crystal structure is designing the initial constructs. Recent advances in high-throughput cloning techniques, affinity purification tags and crystallization robots have however allowed for multiple constructs to be tested rapidly in parallel. 

Several researchers have exploited the wealth of crystal structures of kinase inhibitor complexes to build protein-based pharmacophores for this target class (20). Aronov and Murcko (21) considered the crystal structures of four promiscuous kinase inhibitors, i.e., inhibitors with an affinity of at least 2 pM on five representative kinases. After aligning the structures of the four proteins, they assigned pharmacophoric features to the bound inhibitors, which led to the identification of five clusters of pharmacophoric features. These served to define a five point pharmacophore for kinase frequent hitters (Fig. la). This pharmacophore could discriminate frequent hitters from selective kinase inhibitors. Recently, McGregor (22) aligned 220 kinase crystal structures from the PDB and assigned one of seven possible pharmacophoric types to each atom of the bound ligands. 

Baroni applied this protocol to compare the ATP sites of 23 kinase crystal structures, covering four kinase subfamilies (60). The resulting model could separate the four kinase subfamilies. 

Figure 2.16 Subset of overlayed kinase crystal structures.

The first technique is very intuitive. Out of the few proteins that could be crystallized in a number of different conformations, adenylate kinase is probably the best-studied example. By combining nine observed crystal structures and interpolating between them, a movie was constructed that visualized a hypothetical path of its hinge-bending transition (jVonrhein et al. 1995]). 

We have previously calculated conformational free energy differences for a well-suited model system, the catalytic subunit of cAMP-dependent protein kinase (cAPK), which is the best characterized member of the protein kinase family. It has been crystallized in three different conformations and our main focus was on how ligand binding shifts the equilibrium among these ([Helms and McCammon 1997]). As an example using state-of-the-art computational techniques, we summarize the main conclusions of this study and discuss a variety of methods that may be used to extend this study into the dynamic regime of protein domain motion. 

The catalytic subunit of cAPK contains two domains connected by a peptide linker. ATP binds in a deep cleft between the two domains. Presently, crystal structures showed cAPK in three different conformations, (1) in a closed conformation in the ternary complex with ATP or other tight-binding ligands and a peptide inhibitor PKI(5-24), (2) in an intermediate conformation in the binary complex with adenosine, and (3) in an open conformation in the binary complex of mammalian cAPK with PKI(5-24). Fig.l shows a superposition of the three protein kinase configurations to visualize the type of conformational movement. 

As a template for an intermediate conformation of protein kinase, the crystal structure of the binary complex of cAPK with adenosine (Ibkx.pdb in the Protein Data Bank) was used. As templates for open conformations... 

Fig. 1. Superposition of three crystal structures of cAMP-dependent protein kinase that show the protein in a closed conformation (straight line), in an intermediate conformation (dashed line), and in an open conformation (broken line). The structures were superimposed on the large lobe. In three locations, arrows identify corresponding amino acid positions in the small lobe.

Fig. 2. Conformational free energy of closed, intermediate and open protein kinase conformations. cAPK indicates the unbound form of cAMP-dependent protein kinase, cAPKiATP the binary complex of cAPK with ATP, cAPKiPKP the binary complex of cAPK with the peptide inhibitor PKI(5-24), and cAPK PKI ATP the ternary complex of cAPK with ATP and PKI(5-24). Shown are averaged values for the three crystal structures lATP.pdb, ICDKA.pdb, and ICDKB.pdb. All values have been normalized with respect to the free energy of the closed conformations.

The spatial and steric requirements for high affinity binding to protein kinase C (PKC), a macromolecule that has not yet been crystallized, were determined. Protein kinase C plays a critical role in cellular signal transduction and is in part responsible for cell differentiation. PKC was identified as the macromolecular target for the potent tumor-promoting phorbol esters (25). The natural agonists for PKC are diacylglycerols (DAG) (26). The arrows denote possible sites of interaction. 

De Bondt, H.E., et al. Crystal structure of cyclin-depen-dent kinase 2. Nature 363 595-602, 1993. 

C-terminal tail (Tyr 527 in c-Src). Phosphorylation of Tyr 419 activates the kinase phosphorylation of Tyr 527 inhibits it. Crystal structures of a fragment containing the last four domains of two members of this family were reported simultaneously in 1997—cellular Src by the group of Stephen Harrison and Hck by the group of John Kuriyan. The two structures are very similar, as expected since the 440 residue polypeptide chains have 60% sequence identity. The crucial C-proximal tyrosine that inhibits the activity of the kinases was phosphorylated in both cases the activation loop was not. 

Sicheri, R, Moarefi, 1., Kuriyan, J. Crystal structure of the Src family tyrosine kinase Hck. Nature 385 602-609, 1997. 

Figure 5.9 Models of hexo-kinase in space-filling and wireframe formats, showing the cleft that contains the active site where substrate binding and reaction catalysis occur. At the bottom is an X-ray crystal structure of the enzyme active site, showing the positions of both glucose and ADP as well as a lysine amino acid that acts as a base to deprotonate glucose.

Fluorid ions stimulate bone formation by a direct mitogenic effect on osteoblasts mediated via protein kinase activation and other pathways. Further to these cellular effects, fluorides alter hydroxyapatite crystals in the bone matrix. In low doses, fluorides induce lamellar bone, while at higher doses abnormal woven bone with inferior quality is formed. The effect of fluorides on normal and abnormal (e.g. osteoporotic) bone therefore depends on the dose administered. 

The Sema domain consisting of about 500 amino acids is characterized by highly conserved cysteine residues that form intramolecular disulfide bonds. Crystal structures have revealed that the Sema domain folds in the manner of the (3 propeller topology which is also found in integrins or the low-density lipoprotein (LDL) receptors. Sema domains are found in semaphorins, plexins and in the receptor tyrosine kinases Met and Ron. 

Molecular insight into the protein conformation states of Src kinase has been revealed in a series of x-ray crystal structures of the Src SH3-SH2-kinase domain that depict Src in its inactive conformation [7]. This form maintains a closed structure, in which the tyrosine-phosphorylated (Tyr527) C-terminal tail is bound to the SH2 domain (Fig. 2). The x-ray data also reveal binding of the SH3 domain to the SH2-kinase linker [adopts a polyproline type II (PP II) helical conformation], providing additional intramolecular interactions to stabilize the inactive conformation. Collectively, these interactions cause structural changes within the catalytic domain of the protein to compromise access of substrates to the catalytic site and its associated activity. Significantly, these x-ray structures provided the first direct evidence that the SH2 domain plays a key role in the self-regulation of Src. 

---