The Structural Basis of Familial Parkinson’s Disease Kateryna Zorych The Structural Basis of Familial Parkinson’s Disease Parkinson’s disease is characterized by tremor, stiffness, and slowing movement due to the reduced levels of dopamine. Additional to the motor skills impairment, Parkinson disease is often accompanied by depression, anxiety, and apathy resulting from the loss of the essential neurotransmitter dopamine. Leucine-Rich Repeat Kinase 2 (LRRK2), also known as dardarin, is a protein linked to Parkinson disease. There are four LRRK2 gene variants commonly found in patients with Parkinson’s disease (about one-third of the cases) and is associated with type 8 Parkinson’s disease.

The Structure of Leucine-Rich Repeat Kinase 2 The LRRK2 protein possesses an ankryin repeat region, a leucine-rich repeat (LRR) domain, a kinase domain, a DFG-like motif, a RAS domain, a GTPase domain, a MLK-like domain, and a WD40 domain. The protein is present largely in the cytoplasm but also associates with the mitochondrial outer membrane. Here we present the structure of the Ras of complex proteins (ROC) domain that may act as a GTPase to regulate its protein kinase activity [1].

ROC Domain of The LRRK2 Forms A Dimeric GTPase GDP GDP Mg2+ Mg2+ Chain B Chain A

Surface representation highlighting the GDP-Mg2+ binding pocket on the surface of the dimer that is contributed from both monomers. Mg2+ Binding Pocket GDP Binding Pocket GDP Mg2+

Structural basis of PD-associated mutations in ROC: Mutations at the residues important for dimerization or GTPase formation alter GTPase activity, resulting in pathological consequences. Here, the importance of the residue Arginine R1441 is explored. The arginine-to-glycine substitution at the residue is commonly found in Parkinson’s disease patients of Basque ancestry, [2] while the arginine-to-cysteine mutation is implicated in autosomal dominant Parkinson’s [3]. Many other Parkinson’s-related LRRK2 mutations have been identified. A Drosophila model expressing the human LRRK2 G2019S mutation in neuronal cells showed adult-onset loss of dopaminergic neurons, locomotor dysfunction, and early mortality. Treatment of mutant flies with L-dopa improved locomotor impairment but did not prevent the loss of dopaminergic cells. [4]

The Hydrophobic Zipper at The Dimer Interface is Essential to Proper LRRK2 Function Arginine R1441 and tryptophan W1434 from one monomer (chain A, highlighted in Red) together with phenylalanine F1401 and proline P1406 from the other monomer (chain B, highlighted in Cyan) stack on each other alternately, forming a hydrophobic zipper at the dimer interface [1]. The guanidinium group of R1441 is hydrogen-bonded with the backbone carbonyl oxygen of F1401 and the hydroxyl group of T1404.

Arginine R1441 Interacting with Neighboring Residues H-Bonding Interactions F1401 P1406 R1441

A Proposed Mechanism for the ROC Domain of LRRK2 Regulating Its Kinase Function LRRK2 is an interesting kinase because its two enzymatic domains within a single polypeptide communicate to control activity [5, 6], as the kinase activity of LRRK2 is stimulated upon GTP binding to ROC [7, 8]. Although the mechanism of GTP regulation of kinase activity is not fully understood, it has been postulated that ROC regulates the kinase activity by alternating its conformations through a GTP-bound (active) and GDP-bound (inactive) cycle, which suggests that loss of binding of GTP or increasing turnover of GTP to GDP is likely to result in lowered kinase activity. [1]

Juvenile Parkinsonism: a Devastating Disease Juvenile parkinsonism is defined by its early onset, appearing in patients younger than 40. Familial autosomal recessive Parkinson’s identified in a Japanese family. The defining features of the disease were unusually early onset, generally at adolescence, short lifespan, and the absence of Lewy bodies in the brain at autopsy. [ref 1a] Another Japanese research group published a study of 12 Japanese families, in 11 of which the individuals affected with Parkinson’s were children from consanguineous marriages. In these families, the mean age of parkinsonism onset was 27. Patients responded to levodopa treatment but presented with dopa-induced dyskinesias and wearing-off phenomena [ref 2a] Yet another study identified a possibly new type of early-onset Parkinson’s disease in which the patients did not respond to levodopa but instead showed improvement upon treatment with trihexyphenidyl [ref 3a]

Inheritance Of Juvenile Parkinson’s Disease Juvenile Parkinson’s has been associated with a mutant Park2 gene, found on chromosome 6. The mutations are recessive, with homozygous mutations resulting in juvenile Parkinson’s while the heterozygous mutations are associated with later-onset Parkinson’s. In a group of 15 families from 4 distinct ethnic backgrounds, Jones et al. (1998) [ref 4a] found the locus for autosomal recessive juvenile parkinsonism in all to be mapped to 6q25.2-q27. Notably, the PARK2 gene maps to 6q25.2-q27, the region to which autosomal recessive juvenile parkinsonism maps.

Mutations in Parkin and Familial Parkinson’s Disease The Parkin protein contains an N-terminal ubiquitin-like (UBL) domain and 2 C-terminal RING finger domains that are separated by an in-between ring (IBR) domain. The RING-IBR-RING (RBR) structure is highly conserved and can only be found in eukaryotes. The IBR domain has 2 zinc-binding sites. Zinc binding is required for the correct folding of the IBR domain, which is necessary for proper protein interactions and subsequent ubiquitination.

Parkin IBR domain, the 78 residue stretch (residues M307–S384) comprising the IBR domain depicted in rainbow. The two zinc ions are indicated in the figure. Zn2+ Ions

The Structure of Parkin In-Between Ring (IBR) Domain: According to the NMR structure determination [ref5a], folding of the IBR domain (residues M327–S378) was found to be zinc dependent and the N terminus of the IBR domain, residues E307–E322, was found to be unstructured. The structure shows that the IBR possesses two zinc-binding sites that adopt a dual scissor-like and GAG knuckle-like fold. Furthermore, zinc binding is required for the correct folding of the domain as substitution of zinc coordinating residues (C332S, C365S) causes its global unfolding. The missense T351P mutation found in patients suffering from Autosomal Recessive Juvenile Parkinson’s (ref6a) causes the global unfolding of the IBR domain, whereas the mutation R334C (ref7a) causes some structural rearrangement. In contrast, the protein containing the mutation G328E appears to be properly folded.

Parkin IBR Domain Zinc Coordinating Sites Zinc is essential for proper folding of the IBR domain. Any mutations (deletions/substitutions) of the key residues can abolish zinc coordination and result in a dysfunctional Parkin protein. Zinc Coordinating Site I Zinc Coordinating Site II

Zinc-Coordinating Residues Site I Zn2+ Cysteine

Zinc-Coordinating Residues Site II Zn2+ Histidine Cysteine

Role of Parkin in Protein Degradation: A Link Between Loss-of-Function and Juvenile Parkinsonism The protein Parkin has been identified as an E3 ubiquitin–protein ligase of the ubiquitin-proteosome system that is required to maintain cellular protein quality control by removing misfolded or damaged proteins. Parkin is involved in protein degradation as a ubiquitin-protein ligase collaborating with the ubiquitin-conjugating protein Ubch7. Mutant parkin from patients with autosomal recessive juvenile parkinsonism shows loss of the ubiquitin-protein ligase activity. These findings indicated that accumulation of proteins causes a selective neural cell death without formation of Lewy bodies, which are absent in juvenile Parkinson’s.

The Link Between Parkin and Alpha-Synuclein, Another Protein Involved in Parkinson’s Disease It has been hypothesized that alpha-synuclein and parkin interact functionally and that parkin normally ubiquitinates alpha-synuclein and that this process is altered in PDJ. In contrast to normal parkin, mutant parkin associated with autosomal recessive Parkinson disease failed to bind alpha-Sp22. Consequently, alpha-Sp22 accumulates in a nonubiquitinated form in parkin-deficient Parkinson disease brains. These findings demonstrated a critical biochemical reaction between the two Parkinson disease-linked gene products and suggested that this reaction underlies the accumulation of ubiquitinated alpha-synuclein in conventional Parkinson disease. [ref8a]

Interactions Between Parkin and Leucine-Rich Repeat Kinase 2 It has also been reported that parkin interacts with LRRK2. LRRK2 interacted preferentially with the C-terminal R2 RING finger domain of parkin, and parkin interacted with the COR domain of LRRK2.

Works Cited [1] Proc Natl Acad Sci U S A. 2008 Feb 5;105(5):1499-504. Epub 2008 Jan 29. Structure of the ROC domain from the Parkinson's disease-associated leucine-rich repeat kinase 2 reveals a dimeric GTPase. Deng J, Lewis PA, Greggio E, Sluch E, Beilina A, Cookson MR. Department of Biochemistry and Molecular Biology, Oklahoma State University [2] Paisan-Ruiz, C.; Jain, S.; Evans, E. W.; Gilks, W. P.; Simon, J.; van der Brug, M.; Lopez de Munain, A.; Aparicio, S.; Martinez Gil, A.; Khan, N.; Johnson, J.; Martinez, J. R.; and 9 others : Cloning of the gene containing mutations that cause PARK8-linked Parkinson's disease. Neuron 44: 595-600, 2004. PubMed ID : 15541308 [3] Zimprich, A.; Biskup, S.; Leitner, P.; Lichtner, P.; Farrer, M.; Lincoln, S.; Kachergus, J.; Hulihan, M.; Uitti, R. J.; Calne, D. B.; Stoessl, A. J.; Pfeiffer, R. F.; Patenge, N.; Carballo Carbajal, I.; Vieregge, P.; Asmus, F.; Muller-Myhsok, B.; Dickson, D. W.; Meitinger, T.; Strom, T. M.; Wszolek, Z. K.; Gasser, T. : Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology. Neuron 44: 601-607, 2004. PubMed ID : 15541309 [4] Liu, Z.; Wang, X.; Yu, Y.; Li, X.; Wang, T.; Jiang, H.; Ren, Q.; Jiao, Y.; Sawa, A.; Moran, T.; Ross, C. A.; Montell, C.; Smith, W. W. : A Drosophila model for LRRK2-linked parkinsonism. Proc. Nat. Acad. Sci. 105: 2693-2698, 2008. PubMed ID : 18258746 [5] Marin I (2006) The Parkinson disease gene LRRK2: Evolutionary and structural insights. Mol Biol Evol 23:2423–2433. [6] Mata IF, Wedemeyer WJ, Farrer MJ, Taylor JP, Gallo KA (2006) LRRK2 in Parkinson's disease: protein domains and functional insights. Trends Neurosci 29:286–293 [7] Korr D, et al.(2006) LRRK1 protein kinase activity is stimulated upon binding of GTP to its Roc domain. Cell Signal 18:910–920. [8] Ito G, et al. (2007) GTP binding is essential to the protein kinase activity of LRRK2, a causative gene product for familial Parkinson's disease. Biochemistry 46:1380–1388. [9] Smith WW, Pei Z, Jiang H, Moore DJ, Liang Y, West AB, Dawson VL, Dawson TM, Ross CA (December 2005). "Leucine-rich repeat kinase 2 (LRRK2) interacts with parkin, and mutant LRRK2 induces neuronal degeneration". Proc. Natl. Acad. Sci. U.S.A. 102 (51): 18676–81

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