Eukaryotic Protein Kinase Catalytic Domain

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This page, as it appeared on December 12, 2013, was featured in this article in the journal Biochemistry and Molecular Biology Education.

Contents

Introduction

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.

Tour of Structural Features

The tour in this scrollable section 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.

Twelve Conserved Subdomains

The crystal structure 1atp contains the mouse PKA catalytic (C) subunit (blue cartoon), inhibitor protein PKI (yellow cartoon), the ATP analog ANP (CPK wireframe), and two manganese ions (green spheres). In addition to the protein kinase catalytic domain (residues 43-297), the C subunit contains amino-terminal (residues 1-43) and carboxy-terminal (residues 298-350) sequences. The still image of the model shows the protein kinase fold of catalytic domains of eukaryotic protein kinases, which comprises a small lobe and a large lobe (seen at the top and bottom of the model, respectively) with a catalytic cleft, marked by the bound ANP 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 ANP, and some include the inhibitor peptide to illustrate kinase/substrate interactions.

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

Subdomain II contains an

invariant lysine (ball and stick) that interacts with the phosphates of ATP.

Subdomain III 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 nearly invariant glutamate (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.

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

Subdomain V 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 ATP binding pocket and also for peptide substrate binding. 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.

Subdomain VIa is a long alpha helix in the large lobe that parallels the alpha helix of subdomain IX.

Subdomain VIb contains the catalytic loop with the conserved motif HRDLKxxN (In PKA the H is a Y, instead). The D of this motif (blue ball and stick) 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.

Subdomain VII contains two beta strands link by the Mg-binding loop with the DFG motif. The Aspartate in this motif (blue ball and stick) 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.

Subdomain VIII contains several important features. The APE motif is located at the carboxyl end of this subdomain and the glutamate (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 phosphothreonine (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 P+1 loop (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.

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

Subdomain X and Subdomain XI 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 Subdomains - Functional 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 activation loop 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 activation loop is positioned 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 "spines" (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. The catalytic spine 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) The regulatory spine 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 "gatekeeper" 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 between the two hydrophobic spines [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.

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

Drag the structure with the mouse to rotate

Active and inactive structures

The kinase structure used in the above tour is that of the active conformation of PKA. 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 that occur during a catalytic cycle and in active and inactive enzymes, use the links below to compare inactive, unphosphorylated PKA 4dfy (activation loop threonine is not phosphorylated), active apo PKA 1j3h, and active PKA in complex with ANP and PKI 1atp (the same structure used above), shown in the left, middle, and right frames, respectively. 4dfy shows the structure of an inactive form of PKA, in which the internal structure is disorganized due to the lack of phosphorylation of threonine 197 in the activation loop. Phosphorylation of this residue is required for formation of hydrogen bonds that are critical for alignment of structures to form the active site. 1j3h and 1atp show the open and closed structures assumed by PKA during the catalytic cycle. Note that some residues in 1j3h and 4dfy are not depicted in the models, because they are disordered and not resolved in the structures.

Click on all three links with same number to compare the indicated features. Legends for each set of scenes are below. To reset the structures, reload the page.

4dfy - apo unphosphorylated PKA, inactive

Drag the structure with the mouse to rotate

4dfy
1. Inactive conformation1. Inactive conformation
2. Disassembled spines
3. Critical structures

1j3h - apo PKA, open conformation

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1j3h
1. Open conformation1. Open conformation
2. Assembled, open spines
3. Critical structures

1atp - PKA (blue) with ATP and PKI (yellow); closed and active

Drag the structure with the mouse to rotate

1atp
1. Closed, active conformation1. Closed, active conformation
2. Assembled, closed spines
3. Critical structures

Scene legends
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. (The activation loop of the unphosphorylated PKA is disordered, and thus not represented in the crystal structure.) Note the difference in distance and alignment of these pairs of residues. The small lobe is rotated 18° relative to the active conformation. 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. The spines are assembled in the closed and open active kinases (left and middle scenes), but disorganized in the inactive kinase (right).

3. These scenes show the alignments of structures critical for activity. The yellow Cα-trace is the DFG-activation loop sequence, the blue trace is the catalytic loop, and the orchid trace is the C-helix (subdomain III). In ball and stick are residues critical for catalytic activity: yellow is the D in DFG, which binds Mg2+; blue is the D in the YRDKLPEN, which is the catalytic base; cyan is the invariant K of subdomain II, which binds the phosphates of ATP; and orchid is the invariant E of subdomain III. The positions needed for catalysis can be seen in the closed, active kinase (left). The two D's and K are pointing toward ANP, and the E is bound to the K. The latter pulls the C-helix into position. In the open structure (middle) the elements of the large lobe are in place but the K of the small lobe is far away from the ANP binding site. Upon ATP binding the K interacts with the phosphates and the two lobes close. The view of the inactive structure (right) is oriented so that the backbones of the catalytic loop (blue) and ends of the activation loop (yellow) are positioned like those in the other two structures. The other residues of the activation loop are not shown because they were not resolved in the crystal structure because of their flexibility. The side chain of the D in the catalytic loop (blue ball and stick)points away from the ATP binding pocket, and the C-helix is rotated upward. The assembly of these elements depends on the phosphorylation of threonine 197 in the activation loop. The phosphate of the residue forms five critical bonds that align the active site structures[14].

Regulation of Protein Kinase Activity

There are a variety of ways that the activity of protein kinases are regulated. Here are a few examples. Some are regulated via phosphorylation of residue(s) in the activation loop by either an upstream protein kinase (such as mitogen-activated protein kinase phosphorylation by MAPKK) or by autophosphosphorylation stimulated by the binding of a ligand (such as the insulin receptor kinase[15]). Others are activated by binding with other proteins, which brings the kinase into the active conformation. The PKA C subunit, having been constitutively phosphorylated by an upstream kinase, is active when released from a complex with the regulatory subunit upon the binding of cAMP (see cAMP-dependent protein kinase). Calcium-dependent protein kinase has calcium-binding domain that blocks the active site in the absence of calcium[16]. Upon binding calcium the latter domain undergoes a dramatic conformational change and it moves to a binding site that is on opposite side of the kinase, thus unblocking the catalytic cleft.

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. ↑ 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
  14. ↑ Steichen JM, Kuchinskas M, Keshwani MM, Yang J, Adams JA, Taylor SS. Structural basis for the regulation of protein kinase A by activation loop phosphorylation. J Biol Chem. 2012 Feb 10. PMID:22334660 doi:10.1074/jbc.M111.335091
  15. ↑ Hubbard SR, Wei L, Ellis L, Hendrickson WA. Crystal structure of the tyrosine kinase domain of the human insulin receptor. Nature. 1994 Dec 22-29;372(6508):746-54. PMID:7997262 doi:http://dx.doi.org/10.1038/372746a0
  16. ↑ Wernimont AK, Artz JD, Finerty P Jr, Lin YH, Amani M, Allali-Hassani A, Senisterra G, Vedadi M, Tempel W, Mackenzie F, Chau I, Lourido S, Sibley LD, Hui R. Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium. Nat Struct Mol Biol. 2010 May;17(5):596-601. Epub 2010 May 2. PMID:20436473 doi:10.1038/nsmb.1795

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