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<StructureSection load='4yzm' size='340' side='right' caption='Cocrystal 3D structure of Roco4 kinase and LRRK2-IN-1' scene=''>
<StructureSection load='4yzm' size='340' side='right' caption='Cocrystal 3D structure of Roco4 kinase and LRRK2-IN-1' scene=''>
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Some studies to find a treatment for the Parkinson's disease are focused on LRRK2. Indeed, mutations in LRRK2, which increases its kinase activity, are found in case of Parkinson’s disease. Thus, a kinase inhibitor for LRRK2 would be an interesting thetapeutic target.
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Some treatment targets for the Parkinson's disease are focused on LRRK2. Indeed, mutations increasing its kinase activity are found in both case of the Parkinson’s disease. Thus, a kinase inhibitor for LRRK2 would be an interesting thetapeutic target.<ref name="pnas">doi: 10.1073/pnas.1203223109</ref>
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Thanks to the similarity between LRRK2 and Roco4 from the ''Dictyostelium'', Roco4 is used in studies with a view to finding that inhibitor. One of the candidates to inhibit this activity is <scene name='75/751216/Lrrk2-in-1/3'>LRRK2-IN-1</scene>.<ref name="Bernd">doi: 10.1021/jm5018779</ref>
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Thanks to the similarities between LRRK2 and the ''Dictyostelium'' Roco4, Roco4 is used in studies with a view to find that inhibitor. One of the candidates to inhibit this activity is <scene name='75/751216/Lrrk2-in-1/3'>LRRK2-IN-1</scene>.<ref name="Bernd">doi: 10.1021/jm5018779</ref><ref name="pnas"/><ref name="Wouter">doi: 10.1128/EC.00366-09</ref>
== Humanized Roco4 ==
== Humanized Roco4 ==
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Roco proteins are serine/threonine specific kinases. This family consists of multidomain Ras-GTPases. Roco4 is 193 kDa and is identified as a key protein for proper stalk cell formation. Between the ''Dictyostelium'' Roco genes and LRRK genes, there are many structural similarities, which are due to independant acquisitions of distantly related protein kinase domain.
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Roco proteins are serine/threonine specific kinases. This family consists of multidomain Ras-GTPases. Roco4 has a MW of 193 kDa and is identified as a key protein for proper stalk cell formation. Between the ''Dictyostelium'' Roco genes and LRRK genes, there are many structural similarities, which are due to independant acquisitions of distantly related protein kinase domain.<ref name="Wouter"/>
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The characteristics of roco protein family, are a conserved core, consisting of a Ras-like GTPase domain called ROC (Ras of Complex proteins) and a COR domain (C-terminal of ROC), a C-terminal <scene name='75/751216/Kinase_domain/1'>kinase domain</scene> and several N-terminal leucine rich repeats (LRR). Roco4 possesses one more domain : a C-terminal WD40 repeats.<ref name="Bernd2"/>
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The characteristics of roco protein family are a conserved core, consisting of a Ras-like GTPase domain called ROC (Ras of Complex proteins) and a COR domain (C-terminal of ROC), a C-terminal <scene name='75/751216/Kinase_domain/1'>kinase domain</scene> and several N-terminal leucine rich repeats (LRR). Roco4 possesses one more domain : a C-terminal WD40 repeats.<ref name="Bernd2"/><ref name="Mills">doi: 10.1002/humu.22515</ref><ref name="Wouter"/>
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[[Image:Roco4.jpg|thumb| Linear structure of Roco4 <ref name="Bernd2">doi: 10.3389/fnmol.2014.00032</ref>|center|upright=2,5]]
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[[Image:Roco4.jpg|thumb| Linear structure of Roco4 <ref name="Bernd2">doi: 10.3389/fnmol.2014.00032</ref><ref name="Wouter"/>|center|upright=2,5]]
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The ROC domain possesses five G motifs that are required for guanine nucleotid binding. This domain presents some similarities with the proteins of the ras family. The COR and the ROC domains forms an inseperable tandem, a 300-400 long stretch of amino acids with no significant homology to other described domains. <ref name="Bernd2"/>
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The ROC domain possesses five G motifs that are required for guanine nucleotid binding. This domain presents some similarities with the proteins of the ras family. The COR and the ROC domains forms an inseperable tandem, a 300-400 long stretch of amino acids with no significant homology to other described domains. <ref name="Bernd2"/><ref name="Wouter"/>
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The Roco4 kinase structure consists of a canonical, two-lobed kinase structure, with an adenine nucleotide bound in the conventional <scene name='75/751216/Atp_binding_pocket/1'>ATP-binding pocket</scene>. It contains the conserved alphaC-helix and an anti-parallel beta sheets in the smaller N-terminal lobe. Other Alpha-helices and the activation loop with the conserved N-terminal DFG motif are localized in the bigger C-terminal lobe.<ref name="Bernd2"/>
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The Roco4 kinase structure consists of a canonical, two-lobed kinase structure, with an adenine nucleotide bound in the conventional <scene name='75/751216/Atp_binding_pocket/1'>ATP-binding pocket</scene>. It contains the conserved αC-helix and an anti-parallel β sheet in the smaller N-terminal lobe. Others α-helix and the activation loop with the conserved N-terminal DFG motif are localized in the bigger C-terminal lobe.<ref name="Bernd2"/><ref name="Wouter"/>
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The activation loop and alphaC-helix together form the catalytic site of the kinase, an <scene name='75/751216/Atp_binding_pocket/1'>ATP binding site</scene> formed by a cleft between the two lobes.
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The activation loop and αC-helix together form the catalytic site of the kinase, an <scene name='75/751216/Atp_binding_pocket/1'>ATP binding site</scene> formed by a cleft between the two lobes.
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For catalysis, the formation of a polar contact is essential. This polar contact takes place between Roco4 <scene name='75/751216/1055/2'>Lys1055</scene> from the beta3-strand and the Glu1078 from the alphaC-helix. The amino acids Asp makes contact with all three ATP phosphates either directly or via coordination of a <scene name='75/751216/Mg/1'>magnesium ion</scene>. Moreover, the amino acid Phe makes hydrophobic contacts to the alphaC-helix and the HxD motif, and leads for the correct positioning of the DFG motif. <ref name="Bernd2"/>
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For catalysis, the formation of a polar contact is essential. This polar contact takes place between Roco4 <scene name='75/751216/1055/2'>Lys1055</scene> from the β3-strand and the <scene name='75/751216/1078/1'>Glu1078</scene> from the αC-helix. The amino acid Asp makes contact with all three ATP phosphates either directly or via coordination of a <scene name='75/751216/Mg/1'>magnesium ion</scene>. Moreover, the amino acid Phe makes hydrophobic contacts to the αC-helix and the HxD motif, and leads for the correct positioning of the DFG motif. <ref name="Bernd2"/><ref name="bba">doi: 10.1016/j.bbapap.2016.12.001</ref>
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Roco4 has two conformation, an active conformation and an inactive conformation. These conformations depend of the conformation of the DFG motif : a DFG-in (active) and a DFG-out (inactive) conformation. Therefore, in the structure of active Roco4 kinase, the activation loop is visible and ordered. In contrast, in the structure of inactive Roco4 kinase, the activation loop is not visible.<ref name="Huse">doi: 10.1016/S0092-8674(02)00741-9</ref> <ref name="Taylor">doi: 10.1016/j.tibs.2010.09.006</ref>
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Roco4 has two conformations, an active conformation and an inactive conformation. These conformations depend of the conformation of the DFG motif : a DFG-in (active) and a DFG-out (inactive) conformation. Therefore, in the structure of active Roco4 kinase, the activation loop is visible and ordered. In contrast, in the structure of inactive Roco4 kinase, the activation loop is not visible.<ref name="Huse">doi: 10.1016/S0092-8674(02)00741-9</ref><ref name="Taylor">doi: 10.1016/j.tibs.2010.09.006</ref>
In most kinases, there is a mechanism to switch from an inactive to an active state.
In most kinases, there is a mechanism to switch from an inactive to an active state.
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This involves autophosphorylation of some residues in the activation loop. . Autophosphorylation not only results in the reorientation of the activation loop, but often also alters ATP binding and/or interaction with substrates.<ref name="Huse"/><ref name="Taylor"/> In Roco4 kinase, there are four phosphorylation sites in the activation loop : Ser1181, Ser1184, Ser1187, and Ser1189.<ref name="Bernd"/>
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This involves autophosphorylation of some residues in the activation loop. Autophosphorylation not only results in the reorientation of the activation loop, but often also alters ATP binding and/or interaction with substrates.<ref name="Huse"/><ref name="Taylor"/> In Roco4 kinase, there are four phosphorylation sites in the activation loop : Ser1181, Ser1184, Ser1187, and Ser1189.<ref name="Bernd"/><ref name="Mills"/><ref name="bba"/>
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The structure of ''Dictyostelium'' Roco4 kinase in complex with the LRRK2 inhibitor H1152 allows us to see that Roco4 and other Roco family proteins are essential for the optimization of the current, and identification of new LRRK2 kinase inhibitor. To have a Roco4 protein which have an active site resembling human LRRK2, researchers use a ''Dictyostelium'' Roco4 mutant (F1107L and F1161L) which is called humanized Roco4.<ref name="Bernd"/>
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The structure of ''Dictyostelium'' Roco4 kinase in complex with LRRK2 inhibitors shows that Roco4 and others Roco family proteins are essential for the optimization of current LRRK2 inhibitors, as well as the identification of new ones. In order to work with a protein which have an active site resembling the human LRRK2, researchers use a ''Dictyostelium'' Roco4 mutant (<scene name='75/751216/1107/1'>TF1107L</scene> and <scene name='75/751216/1161/1'>TF1161L</scene>) which is called humanized Roco4.<ref name="Bernd"/>
== LRRK2-IN-1 ==
== LRRK2-IN-1 ==
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[[Image:4K4-270.png|thumb|LRRK2-IN-1 structure (4K4 on PDB website)|upright=1,5]]
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[[Image:4K4-270.png|thumb|LRRK2-IN-1 structure<ref name="4k4"> [https://www3.rcsb.org/ligand/4K4], Retrieved on January 27th 2017.</ref> |upright=1,5]]
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<scene name='75/751216/Lrrk2-in-1/3'>LRRK2-IN-1</scene> is the first identified LRRK2-specific inhibitor, which is now a common tool compound for the LRRK2 research community. LRRK2-IN-1 has a 2-amino-5,11- dimethyl-5H-benzo[e]pyrimido[5,4-b][1,4]diazepine-6(11H)-one scaffold.
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<scene name='75/751216/Lrrk2-in-1/3'>LRRK2-IN-1</scene> is the first identified LRRK2-specific inhibitor, which is know as a common tool compound for the LRRK2 research community. <scene name='75/751216/Lrrk2-in-1/3'>LRRK2-IN-1</scene> has a 2-amino-5,11- dimethyl-5H-benzo[e]pyrimido[5,4-b][1,4]diazepine-6(11H)-one scaffold.
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The function is of LRRK2-In-1 is to dephosphorylate LRRK2 residues Ser910 and Ser935 in the kidney, but not in the brain. This compound is not capable of crossing the blood-brain barrier.
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The function of <scene name='75/751216/Lrrk2-in-1/3'>LRRK2-IN-1</scene> is to dephosphorylate LRRK2 residues Ser910 and Ser935 in the kidney, but not in the brain. This compound is not capable of crossing the blood-brain barrier.
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The structure of LRRK2-In-1 does not stabilize the active conformation. Indeed, the activation loop is poorly resolved indicating that it is flexible. Moreover, it presents a closure of the glycine-rich loop in the inhibitor structure.<ref name="Bernd"/>
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The structure of <scene name='75/751216/Lrrk2-in-1/3'>LRRK2-IN-1</scene> does not stabilize the active conformation. Indeed, the activation loop is poorly resolved indicating that it is flexible. Moreover, it presents a closure of the glycine-rich loop in the inhibitor structure.<ref name="Bernd"/>
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== Humanized Roco4 and LRRK2-IN-1 interaction ==
== Humanized Roco4 and LRRK2-IN-1 interaction ==
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LRRK2-IN-1 makes 2 hydrogen bounds and 24 vander Walls contacts with Roco4. The first hydrogen bound is formed between the backbone carbonyl of <scene name='75/751216/1108/1'>Val1108</scene> and the N24 of the LRRK2-IN-1 with a distance of 2.8 Å. The second is formed between the Nz of <scene name='75/751216/1055/2'>Lys1055</scene> and O40 of LRRK2-IN-1 with a
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<scene name='75/751216/Lrrk2-in-1/3'>LRRK2-IN-1</scene> makes 2 hydrogen bounds and 24 Van Der Waals contacts with Roco4. The first hydrogen bound is formed between the backbone carbonyl of <scene name='75/751216/1108/1'>Val1108</scene> and the N24 of the <scene name='75/751216/Lrrk2-in-1/3'>LRRK2-IN-1</scene> with a distance of 2.8 Å. The second is formed between the Nz of <scene name='75/751216/1055/2'>Lys1055</scene> and O40 of LRRK2-IN-1 with a
distance of 3.2 Å. This interaction takes place in the <scene name='75/751216/Atp_binding_pocket/1'>ATP-binding pocket</scene><ref name="Bernd"/>.
distance of 3.2 Å. This interaction takes place in the <scene name='75/751216/Atp_binding_pocket/1'>ATP-binding pocket</scene><ref name="Bernd"/>.
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LRRK2-IN-1 is predicted to be a type 1 inhibitor. That mean that it should only bind the active form of its target and stabilize it. However, a study showed that it can also bind the inactive form of Roco4<ref name="Bernd"/>.
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<scene name='75/751216/Lrrk2-in-1/3'>LRRK2-IN-1</scene> is predicted to be a type 1 inhibitor. That mean that it should only bind the active form of its target and stabilize it. However, a study showed that it can also bind the inactive form of Roco4<ref name="Bernd"/>.
== Relevance ==
== Relevance ==
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LRRK2, for leucine-rich repeat serin/thereonin kinase 2, is a protein from the Roco family of G-proteins. It takes part in divers pathway such as synaptic vesicule trafficking, retrograde trafficking pathway for recycling protein or the CaMKK/AMPK pathway. Its importance comes from the fact that its susspetced to have a role in the phosphorylation of a central protein in the Parkinson’s disease. (Uniprot) Indeed, mutation associated with Parkinson Disease can be found in asmost every domains of LRRK2. For techrapeutic research Rocco4 from the Dictyostelium was mutated, especially in the active site, in order to mime LRRK2.<ref name="Bernd"/>
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LRRK2 is a protein from the Roco family of G-proteins. It is involved in different pathways such as synaptic vesicule trafficking, retrograde trafficking pathway for recycling protein or the CaMKK/AMPK pathway. This protein is very important because it is suspected to have a role in the phosphorylation of a central protein in the Parkinson’s disease.<ref name="uniprot"> [http://www.uniprot.org/uniprot/Q5S007 UniProtKB - Q5S007 (LRRK2_HUMAN)], Retrieved on January 27th 2017.</ref><ref name="Mills"/> Indeed, mutations associated with Parkinson Disease occur in almost every domains of LRRK2. From a therapeutic point of view and in order to study LRRK2 in Parkinson's disease, researchers use the Roco4 protein. Indeed, Roco4 protein comes from the ''Dictyostelium''. It is mutated in its active site to look like LRRK2.<ref name="Bernd"/><ref name="Mills"/><ref name="pnas"/>
== Disease ==
== Disease ==
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The Parkinson’s disease (PD) is a neurodegenerative disorder that is associated with resting termor, bradykinesia, rigidity and postural instability. (Uniprot) This is the second most common neurodegenerative disorder, which is affecting 2% of the population above 65 years<ref name="Bernd"/>.
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The Parkinson’s disease is a neurodegenerative disorder that is associated with resting tremor, bradykinesia, rigidity and postural instability.<ref name="uniprot"/> This is the second most common neurodegenerative disorder, which affects 2% of the population above 65 years.<ref name="Bernd"/><ref name="Mills"/>
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Two types of Parkinson’s disease existe, the heditary or the sporadic also called idiopathic. LRRK2 mutations can be found in almost its every domains for both types. The most important mutation is the G2019S, which is located on the kinase domain. It stabilise the domains, thus leading to an indresed kinase activity of 2 to 4 fold. That’s why a treatment stategy would be to develop a kinase inhibitor in order to counter it.<ref name="Bernd"/>
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There are two types of Parkinson’s disease: the heditary or the sporadic (also called idiopathic) Parkinson. For both types, LRRK2 mutations occur almost in all of its domains. The most important mutation is the G2019S, which is located in the kinase domain. It stabilises the domain, thus leading to an increased kinase activity of 2 to 4 fold. In order to counter this increase, a treatment strategy is to develop a kinase inhibitor.<ref name="Bernd"/><ref name="Mills"/><ref name="pnas"/>
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The use of roco4, permited to learn that the G2019S mutation is the results of an additional hydrogen bound between Ser2019 (Ser1179 in Roco4) and Gln1918 (Arg1077 in Roco4).<ref name="Bernd"/>
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Thanks to the similarities between Roco4 and LRRK2 proteins, the use of Roco4 allows us to learn that the G2019S mutation is the results of an additional hydrogen bound between Ser2019 (Ser1179 in Roco4) and Gln1918 (Arg1077 in Roco4) in LRRK2.<ref name="Bernd"/><ref name="Mills"/>
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However, LRRK2-IN-1, like many LRRK2 inhibitors, presents a lack of selectivity or difficulties to pass the blood-brain barrier. Kinase inhibitors can lead also to kidney and lung abnormality. Without working on these points,LRRK2-IN-1 couldn’t be used as a treatment for the Parkinson’s disease.
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Like many LRRK2 inhibitors, <scene name='75/751216/Lrrk2-in-1/3'>LRRK2-IN-1</scene> presents a lack of selectivity or difficulties to pass the blood-brain barrier. Kinase inhibitors can lead also to kidney and lung abnormality. Without working on these points, <scene name='75/751216/Lrrk2-in-1/3'>LRRK2-IN-1</scene> could not be used as a treatment for the Parkinson’s disease. Furthermore, <scene name='75/751216/Lrrk2-in-1/3'>LRRK2-IN-1</scene> protein is used as a tool for the characterization of the function and the activation mechanism of LRRK2 in order to find new therapeutic targets. <ref name="Bernd"/>
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In the main time, they are used as tools for characterization of the function and activation mechanism of LRRK2 in order to find new therapeutic target. <ref name="Bernd"/>
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Current revision

Humanized Roco4 bound to LRRK2-IN-1

Cocrystal 3D structure of Roco4 kinase and LRRK2-IN-1

Drag the structure with the mouse to rotate

References

  1. ↑ 1.0 1.1 1.2 1.3 Gilsbach BK, Ho FY, Vetter IR, van Haastert PJ, Wittinghofer A, Kortholt A. Roco kinase structures give insights into the mechanism of Parkinson disease-related leucine-rich-repeat kinase 2 mutations. Proc Natl Acad Sci U S A. 2012 Jun 11. PMID:22689969 doi:10.1073/pnas.1203223109
  2. ↑ 2.00 2.01 2.02 2.03 2.04 2.05 2.06 2.07 2.08 2.09 2.10 Gilsbach BK, Messias AC, Ito G, Sattler M, Alessi DR, Wittinghofer A, Kortholt A. Structural Characterization of LRRK2 Inhibitors. J Med Chem. 2015 May 1. PMID:25897865 doi:http://dx.doi.org/10.1021/jm5018779
  3. ↑ 3.0 3.1 3.2 3.3 3.4 3.5 van Egmond WN, van Haastert PJ. Characterization of the Roco protein family in Dictyostelium discoideum. Eukaryot Cell. 2010 May;9(5):751-61. doi: 10.1128/EC.00366-09. Epub 2010 Mar 26. PMID:20348387 doi:http://dx.doi.org/10.1128/EC.00366-09
  4. ↑ 4.0 4.1 4.2 4.3 4.4 Gilsbach BK, Kortholt A. Structural biology of the LRRK2 GTPase and kinase domains: implications for regulation. Front Mol Neurosci. 2014 May 5;7:32. doi: 10.3389/fnmol.2014.00032. eCollection, 2014. PMID:24847205 doi:http://dx.doi.org/10.3389/fnmol.2014.00032
  5. ↑ 5.0 5.1 5.2 5.3 5.4 5.5 5.6 Mills RD, Mulhern TD, Liu F, Culvenor JG, Cheng HC. Prediction of the repeat domain structures and impact of parkinsonism-associated variations on structure and function of all functional domains of leucine-rich repeat kinase 2 (LRRK2). Hum Mutat. 2014 Apr;35(4):395-412. doi: 10.1002/humu.22515. Epub 2014 Feb 24. PMID:24470158 doi:http://dx.doi.org/10.1002/humu.22515
  6. ↑ 6.0 6.1 Liu Z, West AB. The dual enzyme LRRK2 hydrolyzes GTP in both its GTPase and kinase domains in vitro. Biochim Biophys Acta. 2016 Dec 8;1865(3):274-280. doi:, 10.1016/j.bbapap.2016.12.001. PMID:27939437 doi:http://dx.doi.org/10.1016/j.bbapap.2016.12.001
  7. ↑ 7.0 7.1 doi: https://dx.doi.org/10.1016/S0092-8674(02)00741-9
  8. ↑ 8.0 8.1 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
  9. ↑ [1], Retrieved on January 27th 2017.
  10. ↑ 10.0 10.1 UniProtKB - Q5S007 (LRRK2_HUMAN), Retrieved on January 27th 2017.

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Estelle Metzger

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