Probing the N-Terminal β-Sheet Conversion in the Crystal Structure of the Human Prion Protein Bound to a Nanobody

Abstract

Prions are fatal neurodegenerative transmissible agents causing several incurable illnesses in humans and animals. Prion diseases are caused by the structural conversion of the cellular prion protein, PrP(C), into its misfolded oligomeric form, known as prion or PrP(Sc). The canonical human PrP(C) (HuPrP) fold features an unstructured N-terminal part (residues 23-124) and a well-defined C-terminal globular domain (residues 125-231). Compelling evidence indicates that an evolutionary N-terminal conserved motif AGAAAAGA (residues 113-120) plays an important role in the conversion to PrP(Sc). The intrinsic flexibility of the N-terminal has hampered efforts to obtain detailed atomic information on the structural features of this palindromic region. In this study, we crystallized the full-length HuPrP in complex with a nanobody (Nb484) that inhibits prion propagation. In the complex, the prion protein is unstructured from residue 23 to 116. The palindromic motif adopts a stable and fully extended configuration to form a three-stranded antiparallel β-sheet with the β1 and β2 strands, demonstrating that the full-length HuPrP(C) can adopt a more elaborate β0-β1-α1-β2-α2-α3 structural organization than the canonical β1-α1-β2-α2-α3 prion-like fold. From this structure, it appears that the palindromic motif mediates β-enrichment in the PrP(C) monomer as one of the early events in the conversion of PrP(C) into PrP(Sc).

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

“…In vitro, Nb484 is able to slow down the aggregation into fibrils of murine MoPrP(23e230) in the presence of preformed murine PrP Sc seeds; the lag phase of aggregation kinetics is indeed extended by a factor~2 (~75 h versus 30 h in the presence and absence of Nb484, respectively) [125]. Moreover, Nb484 also inhibits the prion propagation in vivo: scrapie infected murine cells (ScGT1) treated with Nb484 show decreased PrP Sc levels compared to non-treated cells, the effect being Nb484 dose-dependent.…”

“…It was chosen among 14 nanobodies because it displayed the highest affinity for recombinant human PrP (HuPrP (23e231) and HuPrP (90e231)) [125]. Its epitope is discontinuous and includes residues 123e125, the b2-a2 loop (residues 164e170) and half of the a2-helix (residues 174e185) (Fig.…”

“…Nb484 was used as a chaperone to crystallise for the first time the full length human PrP (HuPrP C ) and its C-terminal truncated version (residues 90e231) [125]. The X-ray diffracting crystal of both PrP forms in complex with Nb484 revealed unprecedented structural features at the N-terminal of the palindromic sequence.…”

“…In cells treated with 3.5 mM Nb484, no PrP sc could be detected for at least 7 days and even 14 days after removal of the nanobody from the medium. It is not yet clearly determined if Nb484 inhibits the prion replication by steric hindrance or by preventing structural rearrangements that are essential for the formation of amyloidogenic intermediates [125]. The stabilisation of the b2-a2 loop, which is part of the epitope and whose flexibility has been shown to be important for prion infection, may be particularly relevant.…”

Nanobody-derived nanobiotechnology tool kits for diverse biomedical and biotechnology applications

“…2,48 The structural information on the disordered N-terminal prion protein region has also been elucidated by a VHH-inhibiting prion oligomerization, eventually contributing to the understanding of early prion formation. 2,49 Similarly, crystallization process of components of bacterial type 2 secretion system demonstrated that the VHHs could substantially facilitate well-diffracting crystal formation by merely providing additional contact surface to the target proteins. 2,50,51 By an elegantly experimental design, another example of the use of nanobody is to trigger the depletion of antigen via the ubiquitin pathway.…”