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==Eukaryotic Protein Kinase Catalytic Domain== | ==Eukaryotic Protein Kinase Catalytic Domain== | ||
[[Image:1ATP.jpg|left|]] | [[Image:1ATP.jpg|left|size='90']] | ||
Eukaryotic protein kinases are enzymes that transfer a phosphoryl group (-PO<sub>3</sub><sup>2-</sup>) 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<ref>PMID: 9020587</ref>, humans<ref> PMID:12471243</ref> and rice<ref>PMID:17172291</ref> 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. | |||
Eukaryotic protein kinases are enzymes that transfer a phosphoryl group (-PO<sub>3</sub><sup>2-</sup>) 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<ref>PMID: 9020587</ref>, humans<ref> PMID:12471243</ref> and rice<ref>PMID:17172291</ref> 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<ref name='Hanksa'>PMID:3291115</ref><ref name='Hanksb'>PMID: 7768349</ref> to the three-dimensional structure of protein kinase A (also called PKA or [[CAMP-dependent protein kinase]])<ref name = 'Knightona'> PMID:1862342</ref><ref name = 'Knightonb'>PMID: 1862343</ref>. The results described in these classic papers apply to the basic structure of the great range of eukaryotic protein kinases known today. | This first section of this article relates the twelve conserved subdomains recognized in the primary structures of protein kinase catalytic domains<ref name='Hanksa'>PMID:3291115</ref><ref name='Hanksb'>PMID: 7768349</ref> to the three-dimensional structure of protein kinase A (also called PKA or [[CAMP-dependent protein kinase]])<ref name = 'Knightona'> PMID:1862342</ref><ref name = 'Knightonb'>PMID: 1862343</ref>. 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. | 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 | <StructureSection load='1ATP' size='400' side='right' caption='caption='1atp - Protein kinase A catalytic subunit in complex with ATP (wireframe), manganese, and inhibitor peptide PKI' scene='55/555705/Pkaall/1'>The tour in this scrollable section uses [[1atp]]<ref name = 'Knightonb'>PMID: 1862343</ref> as a model to showcase the twelve conserved subdomains defined by Hanks and Hunter<ref name='Hanksb'>PMID: 7768349</ref>. 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 | ===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. | |||
<scene name='55/555705/Subdomaini/1'>Subdomain I</scene> contains two beta strands connected by the glycine-rich ATP-binding loop with the motif <scene name='55/555705/Gxgxxg/1'>GxGxxG</scene> shown in ball and stick. | <scene name='55/555705/Subdomaini/1'>Subdomain I</scene> contains two beta strands connected by the glycine-rich ATP-binding loop with the motif <scene name='55/555705/Gxgxxg/1'>GxGxxG</scene> shown in ball and stick. | ||
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<scene name='55/555705/Subdomainx/1'>Subdomain X</scene> and <scene name='55/555705/Subdomainxi/1'>Subdomain XI</scene> contain three alpha helices (G, H, and I in mamallian PKA) that form the kinase core and which are involved in binding substrate proteins. | <scene name='55/555705/Subdomainx/1'>Subdomain X</scene> and <scene name='55/555705/Subdomainxi/1'>Subdomain XI</scene> 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 | ===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. | 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. | ||
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Two hydrophobic <scene name='55/555705/Both_spines/2'>"spines"</scene> (reviewed by Taylor and Kornev<ref name ="TaylorTIBS"> PMID: 20971646 </ref>) 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. <scene name='55/555705/Spine1/1'> The catalytic spine </scene>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) <scene name='55/555705/Spine2/1'>The regulatory spine</scene> 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. | Two hydrophobic <scene name='55/555705/Both_spines/2'>"spines"</scene> (reviewed by Taylor and Kornev<ref name ="TaylorTIBS"> PMID: 20971646 </ref>) 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. <scene name='55/555705/Spine1/1'> The catalytic spine </scene>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) <scene name='55/555705/Spine2/1'>The regulatory spine</scene> 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 <scene name='55/555705/Gatekeeper-subdomainv/2'>"gatekeeper"</scene> residue<ref name="TaylorTIBS"/> (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<ref> PMID: 15908922 </ref>. 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 <scene name='55/555705/Gatekeeper-spines/2'>between the two hydrophobic spines </scene><ref name="TaylorTIBS"/> (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 <ref name='one'>PMID: 17114285</ref><ref name='two'>PMID: 18794843</ref>. The gatekeeper's interaction with the two spines affects the orientation of the catalytic, magnesium binding, and activation loops. | The <scene name='55/555705/Gatekeeper-subdomainv/2'>"gatekeeper"</scene> residue<ref name="TaylorTIBS"/> (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<ref> PMID: 15908922 </ref>. 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 <scene name='55/555705/Gatekeeper-spines/2'>between the two hydrophobic spines </scene><ref name="TaylorTIBS"/> (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 <ref name='one'>PMID: 17114285</ref><ref name='two'>PMID: 18794843</ref>. The gatekeeper's interaction with the two spines affects the orientation of the catalytic, magnesium binding, and activation loops.</StructureSection> | ||
==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<ref name = "TaylorTIBS"/>. | |||
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. | |||
< | {| | ||
| <applet load='4DFY' size='300' frame='true' align='right' caption='4dfy - apo unphosphorylated PKA, inactive' scene='55/555705/Unphospka/3' /><Br>'''4dfy'''<Br><scene name='55/555705/Unphospka_spacefill/2'>1. Inactive conformation</scene><br><scene name='55/555705/Unphospka_spines/1'>2. Disassembled spines</scene><br><scene name='55/555705/Unphoscritical/2'>3. Critical structures</scene> | |||
| <applet load='1J3H' size='300' frame='true' align='right' caption='1j3h - apo PKA, open conformation' scene='55/555705/Apopka/2' /><Br>'''1j3h''' <Br><scene name='55/555705/Twistedlobes/2'>1. Open conformation</scene><br><scene name='55/555705/Apo_spines/1'>2. Assembled, open spines</scene><br><scene name='55/555705/Apo_critical/2'>3. Critical structures</scene> | |||
| <applet load='1atp' size='300' frame='true' align='right' caption='1atp - PKA with ANP and PKI; closed and active' scene='55/555705/Pkaall/1' /><Br>'''1atp''' <Br><scene name='55/555705/Closedlobes/4'>1. Closed, active conformation</scene><br><scene name='55/555705/Both_spines/2'>2. Assembled, closed spines</scene><Br><scene name='55/555705/Pkacritical/2'>3. Critical structures</scene> | |||
|} | |||
'''Scene legends'''<br/> | |||
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 Mg<sup>2+</sup>; 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<ref>PMID:22334660</ref>. | |||
===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<ref>PMID:7997262</ref>). 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<ref>PMID:20436473</ref>. 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= | =References= | ||
<references/> | <references/> | ||