Structure and Properties of a Complex of α-Synuclein and a Single-Domain Camelid Antibody

Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.
Journal of Molecular Biology (Impact Factor: 4.33). 09/2010; 402(2):326-43. DOI: 10.1016/j.jmb.2010.07.001
Source: PubMed


The aggregation of the intrinsically disordered protein α-synuclein to form fibrillar amyloid structures is intimately associated with a variety of neurological disorders, most notably Parkinson's disease. The molecular mechanism of α-synuclein aggregation and toxicity is not yet understood in any detail, not least because of the paucity of structural probes through which to study the behavior of such a disordered system. Here, we describe an investigation involving a single-domain camelid antibody, NbSyn2, selected by phage display techniques to bind to α-synuclein, including the exploration of its effects on the in vitro aggregation of the protein under a variety of conditions. We show using isothermal calorimetric methods that NbSyn2 binds specifically to monomeric α-synuclein with nanomolar affinity and by means of NMR spectroscopy that it interacts with the four C-terminal residues of the protein. This latter finding is confirmed by the determination of a crystal structure of NbSyn2 bound to a peptide encompassing the nine C-terminal residues of α-synuclein. The NbSyn2:α-synuclein interaction is mediated mainly by side-chain interactions while water molecules cross-link the main-chain atoms of α-synuclein to atoms of NbSyn2, a feature we believe could be important in intrinsically disordered protein interactions more generally. The aggregation behavior of α-synuclein at physiological pH, including the morphology of the resulting fibrillar structures, is remarkably unaffected by the presence of NbSyn2 and indeed we show that NbSyn2 binds strongly to the aggregated as well as to the soluble forms of α-synuclein. These results give strong support to the conjecture that the C-terminal region of the protein is not directly involved in the mechanism of aggregation and suggest that binding of NbSyn2 could be a useful probe for the identification of α-synuclein aggregation in vitro and possibly in vivo.

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Available from: Els Pardon, Sep 30, 2015
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    • "Recently, solution-state NMR studies on the human recPrP (HuPrP) (residues 90–230) have analyzed the structural rearrangements occurring at the HuPrP urea-denatured state converted in a fibrillar amyloid state, showing that within the protein sequence only the C-terminal residues ∼145–223 constitute the rigid fibril core (Kumar et al., 2010). A powerful tool for unveiling local structural rearrangements within intrinsically disordered proteins linked to protein misfolding diseases is nanobody-aided crystallography (De Genst et al., 2010; De Meyer et al., 2014). A recent approach of this technique in the prion field was employed for determining the crystal structure of the full-length HuPrP bound to a nanobody. "
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    ABSTRACT: Transmissible spongiform encephalopathies (TSE) are a heterogeneous group of neurodegenerative disorders. The common feature of these diseases is the pathological conversion of the normal cellular prion protein (PrP(C)) into a β-structure-rich conformer-termed PrP(Sc). The latter can induce a self-perpetuating process leading to amplification and spreading of pathological protein assemblies. Much evidence suggests that PrP(Sc) itself is able to recruit and misfold PrP(C) into the pathological conformation. Recent data have shown that recombinant PrP(C) can be misfolded in vitro and the resulting synthetic conformers are able to induce the conversion of PrP(C) into PrP(Sc)in vivo. In this review we describe the state-of-the-art of the body of literature in this field. In addition, we describe a cell-based assay to test synthetic prions in cells, providing further evidence that synthetic amyloids are able to template conversion of PrP into prion inclusions. Studying prions might help to understand the pathological mechanisms governing other neurodegenerative diseases. Aggregation and deposition of misfolded proteins is a common feature of several neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and other disorders. Although the proteins implicated in each of these diseases differ, they share a common prion mechanism. Recombinant proteins are able to aggregate in vitro into β-rich amyloid fibrils, sharing some features of the aggregates found in the brain. Several studies have reported that intracerebral inoculation of synthetic aggregates lead to unique pathology, which spread progressively to distal brain regions and reduced survival time in animals. Here, we review the prion-like features of different proteins involved in neurodegenerative disorders, such as α-synuclein, superoxide dismutase-1, amyloid-β and tau. Copyright © 2014. Published by Elsevier B.V.
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    • "In addition, we found that mimicking αS phosphorylation increased the number of inclusions per cell. NMR spectroscopy suggests that the enhanced aggregation is due to a direct binding of Rab8a to the C-terminus of αS that has been implicated in interactions with a variety of protein partners (Burre et al., 2010; De Genst et al., 2010; Jensen et al., 1999, 2000; Kawamata et al., 2001; Kim et al., 2002; Yap et al., 2011). Thus, our study provides a link between physiological αS interactions and pathogenic aggregation. "
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    ABSTRACT: Alpha-Synuclein (αS) misfolding is associated with Parkinson's disease (PD) but little is known about the mechanisms underlying αS toxicity. Increasing evidence suggests that defects in membrane transport play an important role in neuronal dysfunction. Here we demonstrate that the GTPase Rab8a interacts with αS in rodent brain. NMR spectroscopy reveals that the C-terminus of αS binds to the functionally important switch region as well as the C-terminal tail of Rab8a. In line with a direct Rab8a/αS interaction, Rab8a enhanced αS aggregation and reduced αS-induced cellular toxicity. In addition, Rab8 - the Drosophila ortholog of Rab8a - ameliorated αS-oligomer specific locomotor impairment and neuron loss in fruit flies. In support of the pathogenic relevance of the αS-Rab8a interaction, phosphorylation of αS at S129 enhanced binding to Rab8a, increased formation of insoluble αS aggregates and reduced cellular toxicity. Our study provides novel mechanistic insights into the interplay of the GTPase Rab8a and αS cytotoxicity, and underscores the therapeutic potential of targeting this interaction.
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    • "Recombinantly-produced VHHs are termed Nanobodies (Nbs). Nbs are easily expressed in large quantities, are soluble, have high thermal stability, and bind the target antigen with the high affinity and specificity typical of conventional antibodies [24]. Due to their small size and protruding antigen-binding complementarity determining region-3 (CDR3) loop, Nbs have the capacity to reach and recognize cryptic, conformational epitopes that are inaccessible to conventional antibodies [25]–[27]. "
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