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=='''LRRK2/Kinase Inhibitors'''==

==Introduction==

<Structure load='4py1' size='300' frame='true' align='right' caption='TYK2 Protein Chains Painted N terminus to C terminus' scene='48/483886/4py1/1'/>

Parkinson’s disease is one of the most common neurodegenerative diseases worldwide. In comparison, Parkinson’s is ranked just below Alzheimer’s disease, with a 2% lifetime risk. This disorder causes neurons in the body to change their structure dramatically and lose function. These changes trigger symptoms that affect a person’s motor skills, which can include tremors, rigid muscles, slowed movement, change of speech, and weakened balance. While Parkinson’s disease is known for its prevalence with motor skills, it can also affect judgment, decision-making, and memory. Currently there are treatments to manage this disease, but a lot of progress needs to be made to find a cure <ref>PMID: 19042040</ref>.

Leucine rich repeat kinase 2 (LRRK2) is a key protein that can be useful in the treatment of Parkinson’s disease, but currently researchers do not fully comprehend how it specifically correlates to treatment. However, the majority of cases have a similar mutation, <font color='blue'> ''G2019S'' </font>, in the <font color='blue'> ''kinase domain'' </font> of LRRK2, which has proven to increase kinase activity. The idea is to detect kinase inhibitors that can reduce the activity of this mutation in hopes that it will lead towards a better treatment. A kinase is an enzyme that activates phosphate groups to transition to a substrate from high-energy molecules. In order to find more information about the kinase crystal structure of LRRK2, tyrosine kinase 2 (<scene name='48/483886/4py1/1'>TYK2</scene>) is used as a model because it is 74% similar in ATP-binding residues to LRRK2 <ref name ="Galatsis">PMID: 25113930</ref>.

Although, the kinase domain does seem the most relevant to the treatment of Parkinson’s disease, the <font color='red'> ''ROC domain'' </font> of LRRK2 has proven to regulate kinase activity as well. It does this by yielding GDP from GTP through alternations in its conformation in a GTP-bound and GDP-bound cycle. The <font color='red'> ''GDP-Mg2+'' </font> ligand in the ROC domain plays an important role in the complex by stabilizing GDP <ref name ="Deng">PMID: 18230735</ref>.

Amino to Carboxy Rainbow (N->C Rainbow) color key for protein chains in TYK2 (pdb code 4py1)

*current model for kinase domain of LRRK2

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{{Template:ColorKey_Amino2CarboxyRainbow}}

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==Overall Structure==

<Structure load='4py1' size='350' frame='true' align='right' caption='TYK2, 4py1, Secondary Structure' scene='48/483886/Tyk2_secondary_structure/1' />

The Overall Structure of TYK2 is shown in the window on the right and may be returned to by clicking <scene name='48/483886/Tyk2_secondary_structure/1'>here</scene>.

*'''Secondary Structure'''

The protein consists primarily of alpha helices. There are 9 <font color='deeppink'> '''complete helices''' </font>and 5 <font color='darkmagenta'> '''small helical-like structures''' </font> in the protein. <scene name='48/483886/Tyk2_secondary_structure_1/5'>Click</scene> to view the <font color='deeppink'> '''alpha helices'''</font> only. Most helices are clustered in one area, with the exception of 1 full helix and a partial helical loop.

Additionally, there are 11 <font color='gold'> '''beta sheets''' </font>that are distributed in 3 groups among the protein. <scene name='48/483886/Tyk2_secondary_structure_1b/2'>Click</scene> to see only the <font color='gold'> '''beta sheets''' </font>. There is a group of 5 relatively large <font color='gold'> '''beta sheets''' </font> adjacent to 2 medium sized beta sheets. These 2 groups make up most of the “beta region” of the protein. There are another 4 small beta sheets on the opposite end of the protein in the “alpha region”.

<scene name='48/483886/Tyk2_secondary_structure_2/5'>Click</scene> to combine the <font color='deeppink'> '''alpha helices''' </font> and the <font color='gold'> '''beta sheets''' </font> and begin building the protein.

Now <scene name='48/483886/Tyk2_secondary_structure_3/1'>Click</scene> to add the connecting loops.

Finally, <scene name='48/483886/Tyk2_secondary_structure_4/1'>Click</scene> to add the Ligand

*'''Polar and Non-polar Groups'''

<scene name='48/483886/Tyk2_hydrophobic_phillic/3'>Click</scene> to view the entire protein with <font color='orchid'> '''polar''' </font> and <font color='darkgray'> '''non-polar''' </font> coloring.

When looking at the <scene name='48/483886/Tyk2_surf_hydrophobic_phillic/1'>Surface Groups only</scene>, you see that most residues are <font color='orchid'> '''polar''' </font> and there are fewer <font color='darkgray'> '''non-polar''' </font>.

Conversely, the <scene name='48/483886/Tyk2_bury_hydrophobic_phillic/1'>Buried Groups</scene> consist almost entirely of <font color='darkgray'> '''non-polar''' </font> groups. Because the non-polar groups are buried, the protein minimizes the amount of interactions between water and hydrophobic groups. Water molecules are free to hydrogen bond and move freely around the surface of the protein. If non-polar groups were not buried, then water would form an unfavorable low entropy cage structure around the protein. Like many proteins, burying hydrophobic groups creates a stable overall structure.

*'''Location of Hinge Structure'''

There is one critical strand that connects the "alpha region" to the "beta region". This <scene name='48/483886/Hinge_1/3'>strand</scene> may act as a <font color='magenta'> hinge </font> to change the angle between the two regions and increase or decrease exposure to the ligand during binding interactions.

==Binding Interactions==

<Structure load='1a84' size='300' frame='true' align='right' caption='TYK2, 4py1, Binding Interactions' scene='48/483886/4py1_binding_final/1' />

As a kinase, LRRK2 catalyzes the transfer of phosphate groups from high-energy, phosphate-donating molecules (ATP, GTP) to specific substrates. In order to do so, LRRK2 binds to and stabilizes the transition state of ATP or GTP to allow the dephosphorylation of these molecules. Kinase recognition of correct substrates is complex and still poorly understood, however specific binding interactions between LRRK2 and several successful kinase inhibitors have been documented.

The <scene name='48/483886/4py1_binding_final/1'>Binding Interaction Scene</scene> to the right highlights the inhibition of the kinase domain of LRRK2 by [http://www.rcsb.org/pdb/ligand/ligandsummary.do?hetId=2YK 6-((2,5-dimethoxyphenyl)sulfanyl)-3-(1-methyl-1H-pyrazol-4-yl)(1,2,4)triazolo(4,3-b)pyridazine] which we will refer to by its PDB code, <b><font color='steelblue'>'''2YK'''</font></b>. This molecule was shown to inhibit the kinase domain of LRRK2 by altering the conformation of the protein. This inhibitor binds to an active site pocket in the middle of LRRK2 <ref name="Galatsis" />. The residues involved in binding interactions are: <font color='skyblue'> ''' LEU 903, VAL 911, GLY 1040, GLU 979, VAL 981 ''' </font><ref name="Galatsis" />.

The binding pose of this molecule within LRRK2 leaves a small section of the <font color='steelblue'> '''ligand''' </font> exposed to solvent. This <font color='gold'> '''exposed area''' </font> available to solvent can be seen in yellow in the green scene to the left. This allows for hydrogen bonding stabilization of this subsection of the ligand that is not directly interacting with residues inside LRRK2<ref name="Galatsis" />.

[[Image:Binding pocket 4py1.jpg|left|300px]]

The image to the left summarizes the binding interactions between LRRK2 and 2YK. The <font color='red'> '''hydrogen bonds''' </font> with solvent at the exposed surface are represented as red spheres. These are circled and indicated with an arrow.

<b>14-3-3 Interactions </b>

14-3-3 proteins are a family of conserved regulatory molecules that are expressed in all eukaryotic cells <ref name ="Bartel">PMID: 24962282</ref>. 14-3-3 proteins have the ability to bind a multitude of functionally diverse signaling proteins, including kinases, phosphatases, and transmembrane receptors<ref name="Bartel" />. Phosphorylation of LRRK2 at <font color='green'>'''Ser910'''</font> and <font color='green'>'''Ser935'''</font> mediates interaction with 14-3-3<ref>PMID: 24942733</ref>. These residues are highlighted in green in the scene.

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==Additional Features==

<Structure load='2zej' size='300' frame='true' align='right' caption='2zej, GTPase dimer in the LRRK2 ROC domain' scene='48/483886/Roc_dimer_basic/1' />

LRRK2 is a very large protein comprised of multiple domains. The most well-known LRRK2 mutation occurs in the kinase domain and plays a role in Parkinson’s disease by increasing LRRK2's kinase activity.

Researchers have since discovered that the <scene name='48/483886/Roc_dimer_basic/1'>ROC domain</scene> also regulates LRRK2’s kinase activity by functioning as a GTPase which switches between an active GTP- and inactive GDP-bound conformation<ref>PMID: 17706965</ref>. The <scene name='48/483886/Roc_dimer_gdp-mg2_sites/6'>active site</scene> for both monomers is a <font color='red'> '''Mg2+''' </font> binding pocket located on the surface of the ROC dimer, each shown with a bound <font color='orange'> '''GDP'''</font>. Although the mechanism is unclear, LRRK2 is believed to exhibit intramolecular regulation: the activity of LRRK2’s kinase domain is stimulated by the active GTP-bound state of its own ROC domain.

<scene name='48/483886/Roc_dimer_1441_and_1371/1'>Two residues</scene> whose mutations are associated with Parkinson’s disease, <font color='FF4500'> '''R1441'''</font> and <font color='FF00FF'> '''I1371'''</font>, are the key players of LRRK2’s GTPase-regulated kinase activity, because they work together to stabilize the ROC dimer <ref name="Deng" />. Specifically, the <font color='9400D3'> '''guanidine group'''</font> of R1441 exhibits essential <scene name='48/483886/Roc_dimer_hydrogenbonding/5'>hydrogen bonding</scene> with the <font color='FF1493'> '''backbone carbonyl oxygen'''</font> of residue F1401 and the <font color='gold'> '''hydroxyl group'''</font> on the other monomer’s nearby alpha helix. Additionally, the <font color='9400D3'> '''guanidine group'''</font>, W1434’s <font color='DB7093'>'''six membered-ring'''</font>, and the side chain rings of <font color='00CED1'>'''F1401'''</font> and <font color='4169E1'>'''P1406'''</font> create a <scene name='48/483886/Roc_dimer_hydrophobicstacks/1'>hydrophobic “zipper”</scene> via stacking interactions. The <font color='blue'>'''I1371'''</font> residues are located within a <scene name='48/483886/Roc_dimer_hydrophobicpocket/5'>hydrophobic pocket</scene> and provide optimal <scene name='48/483886/Roc_dimer_hydrophobicpocket/3'>van der Waals interactions</scene> with the <font color='black'>'''T1404 methyl group'''</font>.

Due to the intricate hydrogen bonding, hydrophobic contacts, and other important interactions contributed by R1441 and I1371, mutations of either residue can easily destabilize the ROC dimer and alter its function. Research demonstrates that these mutations disrupt the hydrolysis of GTP to GDP, prolonging the active GTP-bound state<ref>PMID: 17623048</ref>. Ultimately, this reduction in GTPase function of the mutated ROC dimer increases LRRK2’s kinase activity, a classic marker of Parkinson’s disease.

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==Quiz Question 1==

<Structure load='4py1' size='300' frame='true' align='right' caption='TYK2, 4py1, Hinge Structure' scene='48/483886/Hinge_structure/1' />

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The molecule shown is TYK2. Since the structure of TYK2 is almost identical to the structure of the kinase region in LRRK2, we will use it to show the concept of the <scene name='48/483886/Hinge_structure/1'>hinge structure</scene>.

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The <scene name='48/483886/Hinge_1/3'>hinge</scene> is the only segment that connects the (mainly) <scene name='48/483886/Upper_cap/2'>beta sheet structure</scene> to the (mainly) <scene name='48/483886/Lower_cap/3'>alpha sheet structure</scene>. Various intramolecular interactions between the <font color='lime'>'''upper'''</font> and <font color='seagreen'>'''lower'''</font> segments help the protein maintain its tertiary structure. The strength of the interactions determines the rigidity of the <font color='deeppink'>'''hinge'''</font>. If we were to add several cysteine molecules that were able to form disulfide bonds between the <font color='lime'>'''upper'''</font> and <font color='seagreen'>'''lower'''</font> segments without disrupting the active site, how would the kinase be affected? In your description, include whether the mutations would primarily affect Km or Vmax, how they would affect these kinetic parameters, and give a brief explanation of why.

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==Quiz Question 2==

<Structure load='1a84' size='300' frame='true' align='right' caption='4PY1, LRRK2 Binding Site Residues' scene='48/483886/Binding_pocket/2' />

To the right is a green scene displaying the <scene name='48/483886/Binding_pocket/2'>binding pocket</scene> of LRRK2 with the residues involved in binding highlighted along with the ligand. The oxygen atoms are shown in red and the nitrogen atoms are shown in blue. Below is the molecular structure of the ligand taken from Galatsis<ref name="Galatsis" /> with one of the ends substituted for R. By looking at the molecular structure presented and considering the binding pocket propose three modifications at the R group which you think would increase the ligands ability to inhibit the kinase.

[[Image:Ligand_R1.png|300px|left|thumb|Ligand with R Group Substitution]]

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==See Also==

*[[http://www.rcsb.org/pdb/explore/explore.do?pdbId=2ZEJ 2ZEJ]]

*[[http://proteopedia.org/wiki/index.php/Leucine-rich_repeat Leucine-rich Repeat]]

*[[http://www.rcsb.org/pdb/explore/explore.do?pdbId=4ECN Crystal structure of a leucine-rich repeat protein]]

*[[http://www.rcsb.org/pdb/explore/explore.do?pdbId=2dl9 Leucine-rich repeat-contain protein 4]]

*[[http://proteopedia.org/wiki/index.php/SAM-dependent_methyltransferase]]

==Credits==

Introduction - Megan Greiner

Overall Structure - Nick Barberio

Drug Binding Site - John Vetrano

Additional Features - Nicole Garvin

Quiz Question 1 - Charit Tippareddy

Quiz Question 2 - Peter Kelly

==References==

<references/>