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

Abstract

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.

Camelid single-domain antibody fragments: Uses and prospects to investigate protein misfolding and aggregation, and to treat diseases associated with these phenomena

“…The study of the interaction of NbSyn87 with a series of peptides spanning the aSyn sequence from residues 118 to 140 further indicates that the majority of the epitope recognised by NbSyn87 is located between residues 118 and 131, and that residues 118 and 119 are critical for binding [11]. The crystal structure of NbSyn2, bound to a peptide corresponding to the last 9 residues of aSyn, reveals that only the last four amino acids of aSyn (137e140) are in direct contact with the nanobody forming a linear epitope [27].…”

“…Two nanobodies, specific to aSyn, have been isolated by phage display from the blood of dromedary [27] and llama [11] immunised with the monomeric human Wt-aSyn or the natural amyloidogenic variant A53T-aSyn. These nanobodies are named NbSyn2 and NbSyn87, and they both bind to the soluble form of a- (Fig.…”

“…Isothermal titration calorimetry (ITC) and quartz crystal microbalance (QCM) data indicate that both nanobodies can bind, even simultaneously, to the aSyn amyloid fibrils, indicating that their specific epitopes, encompassing the C-terminal domain of aSyn, are exposed to the solvent in the fibrils [11,27]. An NMR experiment, in which increasing concentrations of 15 N-labelled NbSyn2 were added to the suspension of a-Syn amyloid fibrils, shows that, one average, four molecule of aSyn in the fibril interact with only one molecule of NbSyn2.…”

“…This ratio could be explained by the steric hindrance of NbSyn2, i.e. one molecule of NbSyn2 could prevent the binding of another molecule of NbSyn2 on the nearest a-synuclein C-termini [27]. Another possibility is the fact that the majority of C-terminus residues of a-synuclein are buried within the fibrils.…”

“…Finally, antibodies recognising epitopes that are generic of oligomeric species or amyloid fibrils have also been described [23e26]. Thus, from a mechanistic point of view, the use of these various specific antibodies could in principle allow to map the apparition of each species in time and can allow a detailed comparison of the aggregation pathways under different conditions including events occurring in vivo [27]. Several antibody formats have been used as structural and mechanistic probes to investigate the aggregation process of diverse proteins: whole mice monoclonal antibodies [28], antibody fragments such as Fab [25], scFv [29,30] and V H [31], and recently, the variable domains of heavy-chain antibodies, referred to as nanobodies or V H Hs.…”