Atomic structure of a nanobody-trapped domain-swapped dimer of an amyloidogenic β2-microglobulin variant

Abstract

Atomic-level structural investigation of the key conformational intermediates of amyloidogenesis remains a challenge. Here we demonstrate the utility of nanobodies to trap and characterize intermediates of β2-microglobulin (β2m) amyloidogenesis by X-ray crystallography. For this purpose, we selected five single domain antibodies that block the fibrillogenesis of a proteolytic amyloidogenic fragment of β2m (ΔN6β2m). The crystal structure of ΔN6β2m in complex with one of these nanobodies (Nb24) identifies domain swapping as a plausible mechanism of self-association of this amyloidogenic protein. In the swapped dimer, two extended hinge loops-corresponding to the heptapetide NHVTLSQ that forms amyloid in isolation-are unmasked and fold into a new twostranded antiparallel β-sheet. The β-strands of this sheet are prone to self-associate and stack perpendicular to the direction of the strands to build large intermolecular β-sheets that run parallel to the axis of growing oligomers, providing an elongation mechanism by self-templated growth.crystallization chaperones | amyloid fibrils | prefibrillar intermediates | dialysis-related amyloidosis P eptides and proteins exhibit a common tendency to assemble into highly ordered fibrillar aggregates, whose formation proceeds in a nucleation-dependent manner (1, 2). The full elucidation of the aggregation process requires the identification of all the conformational states and oligomeric structures adopted by the polypeptide chain. Atomic-level structural investigation of the key conformational intermediates of amyloidogenesis remains a challenge. This is due to the nature of the process, which may be described as a dynamic equilibrium between diverse structural species. These intermediates have dissimilar sizes and occur in very uneven amounts and time frames. Fibril formation in vivo usually takes several years, and the intermediate species are short living and highly unstable (2). Here we demonstrate the utility of heavy chain only antibodies derived from camel (3, 4) for the structural investigation of prefibrillar intermediates of β2-microglobulin (β2m) amyloidosis. The antigen-binding site of these antibodies consists of a single domain, referred to as VHH or nanobody (Nb) (4).β2m is a 99-residue soluble protein that adopts the classical seven-stranded β-sandwich immunoglobulin fold and is expressed as a key component of the major histocompatibility class I complex (MHC-I) on the cell surface of all nucleated cells (5, 6). In healthy individuals, excess β2m is degraded and excreted from the bloodstream by the kidney. In patients suffering from renal failure, the β2m concentration increases up to 60-fold (7) leading to the formation of insoluble amyloid fibrils and causing dialysisrelated amyloidosis (DRA) (8). In amyloid deposits extracted from DRA patients, up to 25-30% of the constituting β2m is truncated and lacks the six N-terminal amino acids (ΔN6β2m) (9, 10). The ΔN6-truncated form of β2m readily aggregates and fibrillates at neutral pH (10, 11).The identification...

Self-assembly of functional, amphipathic amyloid monolayers by the fungal hydrophobin EAS

“…Antiparallel arrangements have been predicted to form amyloid zipper structures (39) but to be rare because of sequence constraints (42). An antiparallel arrangement has been seen in fibrils formed by the Iowa mutant of Aβ1-40 under certain conditions (43), and recent models for RNase A, cystatin, and β 2 -microglobulin amyloid fibrils that involve domain swapping suggest that in the more complex situation of amyloid formation by segments within a mostly folded protein, antiparallel sheets may form steric zippers (44)(45)(46). The model we present for EAS rodlets is similar to the antiparallel, face-to-face steric zipper, as proposed by Sawaya and colleagues (39), but the sheets are composed of different sequences.…”

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

“…A series of eight nanobodies has been selected by phage display from the blood of camel and llama immunised with the monomeric human Wt-b2m or DN6b2m; these two proteins were also used for the panning [94]. The affinities of these nanobodies for b2m and its DN6 variant are in the nanomolar range (Fig.…”

“…When incubated at pH 5, 37 C, in the presence of salt, DN6b2m is rapidly (i.e. within 124 h) converted into 'non-amorphous aggregates' that bind thioflavin-T (ThT); after 2 weeks of incubation, the first amyloid fibrils start to grow out of these aggregates while all aggregates are converted into amyloid fibrils after 4 weeks [94]. Remarkably, in the presence of a 12% excess of five of the eight nanobodies, DN6b2m, incubated at 37 C, does not aggregate within 124 h (Fig.…”

“…The five inhibitory nanobodies were tested as co-crystallisation chaperones of DN6b2m but diffracting crystals were obtained only for the complex with Nb24 [94]. Under the conditions of crystallisation of the complex, the DN6b2m alone aggregates within minutes, indicating that the nanobody indeed serves as an efficient crystallisation chaperone.…”

“…Detection of the b2m in the MHC-I at the surface of cells Nb24 does not bind to b2m within the MHC-I complex exposed on the surface of human cells [94]. Thus, this nanobody efficiently blocks the formation of amyloid fibrils without interfering with the biological function of b2m.…”