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Distinct hydration properties of wild-type and familial point mutant A53T of ?-synuclein associated with Parkinson's disease.

ABSTRACT: The propensity of ?-synuclein to form amyloid plays an important role in Parkinson's disease. Three familial mutations, A30P, E46K, and A53T, correlate with Parkinson's disease. Therefore, unraveling the structural effects of these mutations has basic implications in understanding the molecular basis of the disease. Here, we address this issue through comparing details of the hydration of wild-type ?-synuclein and its A53T mutant by a combination of wide-line NMR, differential scanning calorimetry, and molecular dynamics simulations. All three approaches suggest a hydrate shell compatible with a largely disordered state of both proteins. Its fine details, however, are different, with the mutant displaying a somewhat higher level of hydration, suggesting a bias to more open structures, favorable for protein-protein interactions leading to amyloid formation. These differences disappear in the amyloid state, suggesting basically the same surface topology, irrespective of the initial monomeric state.

SUBMITTER: Hazy E

PROVIDER: S-EPMC3207174 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Distinct hydration properties of wild-type and familial point mutant A53T of α-synuclein associated with Parkinson's disease.

Hazy E E Bokor M M Kalmar L L Gelencser A A Kamasa P P Han K-H KH Tompa K K Tompa P P

Biophysical journal 20111101 9

The propensity of α-synuclein to form amyloid plays an important role in Parkinson's disease. Three familial mutations, A30P, E46K, and A53T, correlate with Parkinson's disease. Therefore, unraveling the structural effects of these mutations has basic implications in understanding the molecular basis of the disease. Here, we address this issue through comparing details of the hydration of wild-type α-synuclein and its A53T mutant by a combination of wide-line NMR, differential scanning calorimet ...[more]

PMID: 22067166

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Project description:The A53T genetic missense mutation of the wild-type ?-synuclein (?S) protein was initially identified in Greek and Italian families with familial Parkinson's disease. Detailed understanding of the structures and the changes induced in the wild-type ?S structure by the A53T mutation, as well as establishing the direct relationships between the rapid conformational changes and free energy landscapes of these intrinsically disordered fibrillogenic proteins, helps to enhance our fundamental knowledge and to gain insights into the pathogenic mechanism of Parkinson's disease. We employed extensive parallel tempering molecular dynamics simulations along with thermodynamic calculations to determine the secondary and tertiary structural properties as well as the conformational free energy surfaces of the wild-type and A53T mutant-type ?S proteins in an aqueous solution medium using both implicit and explicit water models. The confined aqueous volume effect in the simulations of disordered proteins using an explicit model for water is addressed for a model disordered protein. We also assessed the stabilities of the residual secondary structure component interconversions in ?S based on free energy calculations at the atomic level with dynamics using our recently developed theoretical strategy. To the best of our knowledge, this study presents the first detailed comparison of the structural properties linked directly to the conformational free energy landscapes of the monomeric wild-type and A53T mutant-type ?-synuclein proteins in an aqueous solution environment. Results demonstrate that the ?-sheet structure is significantly more altered than the helical structure upon A53T mutation of the monomeric wild-type ?S protein in aqueous solution. The ?-sheet content close to the mutation site in the N-terminal region is more abundant while the non-amyloid-? component (NAC) and C-terminal regions show a decrease in ?-sheet abundance upon A53T mutation. Obtained results utilizing our new theoretical strategy show that the residual secondary structure conversion stabilities resulting in ?-helix formation are not significantly affected by the mutation. Interestingly, the residual secondary structure conversion stabilities show that secondary structure conversions resulting in ?-sheet formation are influenced by the A53T mutation and the most stable residual transition yielding ?-sheet occurs directly from the coil structure. Long-range interactions detected between the NAC region and the N- or C-terminal regions of the wild-type ?S disappear upon A53T mutation. The A53T mutant-type ?S structures are thermodynamically more stable than those of the wild-type ?S protein structures in aqueous solution. Overall, the higher propensity of the A53T mutant-type ?S protein to aggregate in comparison to the wild-type ?S protein is related to the increased ?-sheet formation and lack of strong intramolecular long-range interactions in the N-terminal region in comparison to its wild-type form. The specific residual secondary structure component stabilities reported herein provide information helpful for designing and synthesizing small organic molecules that can block the ?-sheet forming residues, which are reactive toward aggregation.

Project description:Background: Mutations in glucocerebrosidase (GBA), a lysosomal enzyme are the most common genetic risk factor for developing Parkinson's disease (PD). We studied how reduced GCase activity affects α-synuclein (α-syn) and its mutants (A30P and A53T) aggregation, neurodegeneration, sleep and locomotor behavior in a fly model of PD. Methods: We developed drosophila with GBA gene knockdown (RNAi) (with reduced GCase activity) that simultaneously expresses either wildtype (WT) or mutants such as A30P or A53T α-syn. Western blot and confocal microscopy were performed to study the α-syn aggregation and neurodegeneration in these flies. We also studied the sleep and locomotor activity of those flies using Drosophila activity monitor (DAM) system. Results: Western blot analysis showed that GBA RNAi A53T α-syn flies (30 days old) had an increased level of Triton insoluble synuclein (that corresponds to α-syn aggregates) compared to corresponding A53T flies without GBA RNAi (control), while mRNA expression of α-syn remained unchanged. Confocal imaging of whole brain staining of 30 days old drosophila showed a statistically significant decrease in neuron numbers in PPL1 cluster in flies expressing α-syn WT, A30P and A53T in the presence GBA RNAi compared to corresponding control. Staining with conformation specific antibody for α-syn aggregates showed an increased number of neurons staining for α-syn aggregates in A53T fly brain with GBA RNAi compared to control A53T flies, thus confirming our protein analysis finding that under decreased GBA enzyme activity, mutant A53T aggregates more than the control A53T without GBA silencing. Sleep analysis revealed decreased total activity in GBA silenced flies expressing mutant A53T compared to both A53T control flies and GBA RNAi flies without synuclein expression. Conclusion: In A53T flies with reduced GCase activity, there is increased α-syn aggregation and dopamine (DA) neuronal loss. This study demonstrates that reduced GCase activity both in the context of heterozygous GBA1 mutation associated with PD and in old age, contribute to increased aggregation of mutant α-syn A53T and exacerbates the phenotype in a fly model of PD.

Dataset Information

Distinct hydration properties of wild-type and familial point mutant A53T of ?-synuclein associated with Parkinson's disease.

Publications

Distinct hydration properties of wild-type and familial point mutant A53T of α-synuclein associated with Parkinson's disease.

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