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Eukaryotic Protein Kinase Catalytic DomainEukaryotic Protein Kinase Catalytic Domain

Eukaryotic protein kinases are enzymes that transfer a phosphoryl group (-PO32-) 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 and responses to the environment. Also, unregulated versions of kinases that arise from tumor-promoting viruses promote cancer in humans. The number of protein kinase genes (and the percentage of the genome) in 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 proteins.

This first section of this article relates the twelve conserved subdomains recognized in the primary structures of protein kinase catalytic domains[4][5] to the three-dimensional structure of protein kinase A (also called PKA or CAMP-dependent protein kinase)[6][7]. The results described in these classic papers apply to the basic structure of the great range of eukaryotic protein kinases known today.

The second section of this article examines functional structures and assemblies of protein kinase catalytic domains and compares active and inactive conformations.

Twelve Conserved SubdomainsTwelve Conserved Subdomains

The following tour uses 1atp[7] as a model to showcase the twelve conserved subdomains defined by Hanks and Hunter[5]. The subdomains are numbered starting at the amino terminal end of the catalytic domain.

The crystal structure 1atp contains the mouse PKA catalytic subunit (blue cartoon), inhibitor protein PKI (yellow cartoon), ATP (CPK wireframe), and two manganese ions (green spheres). The still image of the model 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, respectively), and that the catalytic cleft, marked by the bound ATP molecule, is located between them. The small lobe binds ATP and the large lobe binds the protein substrate, modeled here by the inhibitor peptide PKI. PKI has an alanine substituted for the serine in the phosphorylation motif RRxS, and thus is unable to be phosphorylated. All of the molecular scenes in the tour include ATP, and some include the inhibitor peptide to illustrate kinase/substrate interactions.

1atp - Protein kinase A catalytic subunit in complex with ATP (wireframe), manganese, and inhibitor peptide PKI

Drag the structure with the mouse to rotate

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 that parallels the alpha helix of subdomain IX.

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. 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.

contains two beta strands link by the Mg-binding loop with the DFG motif. The chelates a Mg2+ ion (Mn2+ in the 1atp crystal structure) 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 XI. 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 (blue ball and stick), which interacts with the residue (yellow ball and stick) adjacent to the phosphorylated residue of the peptide substrate (yellow). The "P" residue is the one that is phosphoryated in the substrate, and the "P + 1" residue is the next residue in the sequence.

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

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

Beyond the Conserved SubdomainsBeyond the Conserved Subdomains

Functional units and assembliesFunctional units and assemblies

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 was first described by Taylor and Radzio-Andzelm[8]. It 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, 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. The activation loop takes on a variety of conformations in inactive kinases[9], that disrupt one or all of these conformations.

Two hydrophobic (reviewed by Taylor and Kornev[10]) 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.

The residue[10] (chartreuse spacefill) is a part of subdomain V (blue) and it is located deep in the ATP-binding pocket (Subdomain I with its ATP binding loop are shown in yellow). The size of the gatekeeper residue determines the size of the binding pocket, and it is thus a gatekeeper for which nucleotides, ATP analogs, and inhibitors can bind[11]. In PKA and about 75% of all kinases it is a large residue, such as leucine, phenylalanine or methionine as seen here. In the remaining kinases, especially tyrosine kinases, the residue is larger, such as threonine or valine. The gatekeeper's location is [10] (gatekeeper is chartreuse, catalytic spine is blue, regulatory spine is orchid). Mutation of this residue in some kinases leads to activation of the kinase via enhanced autophosphorylation of the activation loop, and the unregulated kinase activity promotes cancer [12][13]. The gatekeeper's interaction with the two spines affects the orientation of the catalytic, magnesium binding, and activation loops.

Active and inactive structuresActive and inactive structures

The kinase structure used in the above tour is that of the active conformation of PKA. Following is a comparison of this active conformation, with its particular arrangement of structural elements, to the inactive conformation. While active conformations of protein kinases are very similar, there is great variation in the inactive conformations of protein kinases, but all involve misalignment of one or more of the structures, subdomain III (C-helix in PKA) and the catalytic, magnesium binding, and activation loops[10].

To get an idea of the structural differences in active and inactive kinase domains, on the left is PKA in complex with ANP and PKI (1atp], the same structure used above), and on the right is inactive apo-PKA 1j3h.

4dfy - apo unphosphorylated PKA, inactive

Drag the structure with the mouse to rotate

In the left frame:

1j3h - apo PKA, open conformation

Drag the structure with the mouse to rotate

In the right frame:

1atp - PKA with ANP and PKI; closed and active

Drag the structure with the mouse to rotate


In the middle frame:

1. In these scenes the catalytic domains are shown in spacefill, with the large lobe in silver and the small lobe in blue. To aid viewing, The N and C terminal sequences are in cartoon. Stop the rotation and use your mouse to get a good look at the catalytic cleft, which in 1ATP is closed around ANP. Two sets of residues are shown in yellow and red, respectively, to show the degree to which the cleft opens, and the two lobes twist with respect to each other. The yellow residues are Gly52 from the GxGxxG motif and Thr 201 in the activation loop. The red residues are His 87 in subdomain III (the C helix) and phosphorthreonine 197 in the activation loop. Note the difference in distance and alignment of these pairs of residues. In the closed, active conformation His 87 and phosphoThr 197 have an ionic interaction, whereas in the open conformation they are too far away from each other to interact.

2. These scenes show the catalytic spine in blue space fill and the regulatory spine in orchid spacefill.

ReferencesReferences

  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. ↑ 5.0 5.1 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. ↑ 7.0 7.1 Knighton DR, Zheng JH, Ten Eyck LF, Xuong NH, Taylor SS, Sowadski JM. Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science. 1991 Jul 26;253(5018):414-20. PMID:1862343
  8. ↑ Taylor SS, Radzio-Andzelm E. Three protein kinase structures define a common motif. Structure. 1994 May 15;2(5):345-55. PMID:8081750
  9. ↑ Huse M, Kuriyan J. The conformational plasticity of protein kinases. Cell. 2002 May 3;109(3):275-82. PMID:12015977
  10. ↑ 10.0 10.1 10.2 10.3 Taylor SS, Kornev AP. Protein kinases: evolution of dynamic regulatory proteins. Trends Biochem Sci. 2011 Feb;36(2):65-77. doi: 10.1016/j.tibs.2010.09.006. Epub, 2010 Oct 23. PMID:20971646 doi:10.1016/j.tibs.2010.09.006
  11. ↑ Zhang C, Kenski DM, Paulson JL, Bonshtien A, Sessa G, Cross JV, Templeton DJ, Shokat KM. A second-site suppressor strategy for chemical genetic analysis of diverse protein kinases. Nat Methods. 2005 Jun;2(6):435-41. PMID:15908922 doi:10.1038/nmeth764
  12. ↑ Emrick MA, Lee T, Starkey PJ, Mumby MC, Resing KA, Ahn NG. The gatekeeper residue controls autoactivation of ERK2 via a pathway of intramolecular connectivity. Proc Natl Acad Sci U S A. 2006 Nov 28;103(48):18101-6. Epub 2006 Nov 17. PMID:17114285 doi:10.1073/pnas.0608849103
  13. ↑ Azam M, Seeliger MA, Gray NS, Kuriyan J, Daley GQ. Activation of tyrosine kinases by mutation of the gatekeeper threonine. Nat Struct Mol Biol. 2008 Oct;15(10):1109-18. Epub 2008 Sep 14. PMID:18794843 doi:10.1038/nsmb.1486
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