Structure and properties of a complex of alpha-synuclein and a single-domain camelid antibody.
; ; et al
in Journal of Molecular Biology (2010), 402(2), 326-43
The aggregation of the intrinsically disordered protein alpha-synuclein to form fibrillar amyloid structures is intimately associated with a variety of neurological disorders, most notably Parkinson's ... [more ▼]
The aggregation of the intrinsically disordered protein alpha-synuclein to form fibrillar amyloid structures is intimately associated with a variety of neurological disorders, most notably Parkinson's disease. The molecular mechanism of alpha-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 alpha-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 alpha-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 alpha-synuclein. The NbSyn2:alpha-synuclein interaction is mediated mainly by side-chain interactions while water molecules cross-link the main-chain atoms of alpha-synuclein to atoms of NbSyn2, a feature we believe could be important in intrinsically disordered protein interactions more generally. The aggregation behavior of alpha-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 alpha-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 alpha-synuclein aggregation in vitro and possibly in vivo. [less ▲]Detailed reference viewed: 60 (3 ULg)
(1)H, (13)C and (15)N assignments of a camelid nanobody directed against human alpha-synuclein.
; ; et al
in Biomolecular NMR Assignments (2009), 3(2), 231-3
Nanobodies are single chain antibodies that are uniquely produced in Camelidae, e.g. camels and llamas. They have the desirable features of small sizes (Mw < 14 kDa) and high affinities against antigens ... [more ▼]
Nanobodies are single chain antibodies that are uniquely produced in Camelidae, e.g. camels and llamas. They have the desirable features of small sizes (Mw < 14 kDa) and high affinities against antigens (Kd approximately nM), making them ideal as structural probes for biomedically relevant motifs both in vitro and in vivo. We have previously shown that nanobody binding to amyloidogenic human lysozyme variants can effectively inhibit their aggregation, the process that is at the origin of systemic amyloid disease. Here we report the NMR assignments of a new nanobody, termed NbSyn2, which recognises the C-terminus of the intrinsically disordered protein, human alpha-synuclein (aS), whose aberrant self-association is implicated in Parkinson's disease. [less ▲]Detailed reference viewed: 415 (79 ULg)