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Eukaryotic Protein Kinase Catalytic Domain

Eukaryotic protein kinases are enzymes that transfer a phosphoryl group (-PO₃^2-) from adenosine triphosphate (or more rarely from adenosine diphosphate) to the hydroxyl group of serine, threonine, or tyrosine residue of a protein substrate. Phosphorylation of the substrate can affect its activity and/or conformation and, in turn, the physiogy of the cell. Protein kinases act as switches that turn on or off metabolic and signaling pathways, and they play central roles in development, responses to the environment, and in diseases such as cancer. The number of protein kinase genes (and the percentage of the genome) for bakers yeast ^[1], humans ^[2] and rice ^[3] are 113 (2%),518 (2%), and 1429 (5%), respectively. The catalytic domains of these enzymes occur alone or with other functional domains in a single polypetide chain. Protein kinases may be monomeric or multimeric or found in complexes with regulatory protiens.

This article describes the general structure of protein kinase domains. It is based on the analysis of the primary structure of protein kinases by Hanks, Quinn, and Hunter ^[4] in which the amino acid sequences of 65 protein kinases were aligned, and the revised analysis by Hanks and Hunter ^[5], and on the first three-dimensional structure of protein kinase to be published, that of PKA by Knighton et al. ^[6]. The results described in these papers apply to the basic structure of the great range of eukaryotic protein kinases known today.

The crystal structure 1ATP shows that catalytic domains of eukaryotic protein kinases have a small lobe and a large lobe (seen at the top and bottom of the model in the scene, respectively), and the catalytic site is located in a cleft between them. The small lobe binds ATP and the large lobe binds the protein substrate. The crystal structure includes ATP^.2Mg²⁺ and the inhibitor peptide that has an alanine substituted for serine in a substrate's phosphorylation motif RRxS. All of the molecular scenes below include ATP, and some include the inhibitor peptide to illustrate kinase/substrate interactions.

Twelve Conserved Subdomains

Following is a tour of the twelve conserved subdomains (numbered starting at the amino terminal end of the catalytic domain) defined by Hanks and Hunter using 1PKA as a model.

spinqualitylabelsall models 1PKA - Protein kinase A in complex with ATP, magnesium and inhibitor peptide Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

contains two beta strands connected by the glycine-rich ATP-binding loop with the motif shown in ball and stick.

contains an that interacts with the phosphates of ATP.

is an alpha helix (helix C in bovine PKA) that connects to many parts of the kinase, and its orientation is critical for activity. In the active conformation of the kinase the (shown as blue ball and stick) in Subdomain III forms a salt bridge with the invariant lysine of Subdomain II (yellow ball and stick). This salt bridge couples subdomain III to ATP.

contains a beta strand and contributes to the core structure of the small lobe.

contains a hydrophobic beta strand in the small lobe and an alpha helix in the large lobe. The sequence that links these two secondary structures not only links together the small and large lobes of the kinase, but also contributes residues to the and also for . In PKA Glu 127 (blue ball and stick) interacts with both the ribose of ATP and the first Arg (yellow ball and stick) in the phosphorylation motif RRxS of a peptide substrate.

is a long alpha helix in the large lobe.

contains the catalytic loop with the conserved motif HRDLKxxN (In PKA the H is a Y, instead). The is the catalytic base that accepts the hydrogen removed from the hydroxyl group being phosphorylated. CHECK THIS FOR ACCURACY. Note the proximity of the glutamate residue to peptide residue that will be phosphorylated, here represented by an alanine (yellow ball and stick) in the inhibitor peptide. A substrate peptide would contain a serine instead of the alanine, and the hydroxyl group would narrow the gap between the substrate and the glutamate.

, the Mg-binding loop with the DFG motif. The chelates a Mg²⁺ ion that bridges the gamma and beta phosphates of ATP and positions the gamma phosphate for transfer to the substrate.

contains several important features. The APE motif is located at the carboxyl end of this subdomain and the (blue ball and stick) in this motif forms a salt bridge with an arginine (yellow ball and stick) in in Subdomain X1. This salt bridge is critical for forming the stable kinase core and it provides an anchor for the movement of the activation loop (see below). In many protein kinases there is a phosphorylatable residue seven to ten residues upstream of the APE motif. In PKA it is a (blue ball and stick with the phosphate in CPK), which forms an ionic bond with the arginine (yellow ball and stick) in the YRDLKPEN motif of the catalytic loop and helps to position it for catalysis. Kinases that don't have a phosphorylatable residue in this loop often have an acididc residue that can form the salt bridge. Between the phosphorylated residue and the APE motif lies the which interacts with the residue adjacent to the phosphorylated residue of the peptide substrate.

is a very hydrophobic alpha helix (helix F in bovine PKA). It contains and invariant aspartate residue that is discussed below.

and contain three alpha helices (G, H, and I in bovine PKA) that form the kinase core and which are involved in binding substrate proteins.

Beyond the Conserved Subdomains

Functional structures that involve residues from more than one subdomain have been recognized by biochemical and molecular genetic studies coupled with three-dimensional structures of protein kinases.

The (first described by Taylor and Radzio-Andzelm ^[7])are important for the structure of active conformation of protein kinases. They are composed of amino acid residues that are non-contiguous in the primary structure.includes the adenine ring of ATP. In PKA it comprises residues (from top to bottom in the scene) A70, V57, ATP, L173, I174, L172, M128, M231, and L227, and it is directly anchored to amino end of helix F (Subdomain IX) contains residues L106, L95, F185, Y164, and it is anchored to helix F via a hydrogen bond between the invariant aspartate in helix F (yellow ball and stick) and the backbone nitrogen of Y164. This spine is assembled in the active conformation and disorganized in inactive conformations.

==References==

1. ↑ Hunter T, Plowman GD. The protein kinases of budding yeast: six score and more. Trends Biochem Sci. 1997 Jan;22(1):18-22. PMID:9020587
2. ↑ Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S. The protein kinase complement of the human genome. Science. 2002 Dec 6;298(5600):1912-34. PMID:12471243 doi:10.1126/science.1075762
3. ↑ Dardick C, Chen J, Richter T, Ouyang S, Ronald P. The rice kinase database. A phylogenomic database for the rice kinome. Plant Physiol. 2007 Feb;143(2):579-86. Epub 2006 Dec 15. PMID:17172291 doi:10.1104/pp.106.087270
4. ↑ Hanks SK, Quinn AM, Hunter T. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science. 1988 Jul 1;241(4861):42-52. PMID:3291115
5. ↑ Hanks SK, Hunter T. Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J. 1995 May;9(8):576-96. PMID:7768349
6. ↑ Knighton DR, Zheng JH, Ten Eyck LF, Ashford VA, Xuong NH, Taylor SS, Sowadski JM. Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science. 1991 Jul 26;253(5018):407-14. PMID:1862342
7. ↑ ) comprises amino acid residues between the DFG motif in subdomain VII to the APE motif in subdomain VIII. As it's name implies, it is involved in switching the activity of the kinase on and off. When the phosphorylatable residue in subdomain VIII (see above) is phosphorylated, the such that the active site cleft is accessible, and the magnesium loop (DFG motif) and catalytic loop (HRDLKPxxN motif are properly positioned for catalysis, and the P+1 loop can interact with the peptide substrate. Two hydrophobic "spines" (reviewed by Taylor and Kornev <ref> PMID: 20971646 </li></ol></ref>