We report the effects of the interaction of two camelid
antibody fragments, generally called
nanobodies, namely cAb-HuL5 and a stabilized and more aggregation-resistant variant cAb-HuL5G obtained by
protein engineering, on the properties of two amyloidogenic variants of human
lysozyme, I56T and D67H, whose deposition in vital organs including the liver, kidney, and spleen is associated with a familial non-neuropathic systemic
amyloidosis. Both NMR spectroscopy and X-ray crystallographic studies reveal that cAb-HuL5 binds to the α-domain, one of the two lobes of the native
lysozyme structure. The binding of cAb-HuL5/cAb-HuL5G strongly inhibits fibril formation by the amyloidogenic variants; it does not, however, suppress the locally transient cooperative unfolding transitions, characteristic of these variants, in which the β-domain and the C-helix unfold and which represents key early intermediate species in the formation of
amyloid fibrils. Therefore, unlike two other
nanobodies previously described, cAb-HuL5/cAb-HuL5G does not inhibit fibril formation via the restoration of the global cooperativity of the native structure of the
lysozyme variants to that characteristic of the wild-type
protein. Instead, it inhibits a subsequent step in the assembly of the fibrils, involving the unfolding and structural reorganization of the α-domain. These results show that
nanobodies can protect against the formation of pathogenic aggregates at different stages in the structural transition of a
protein from the soluble native state into
amyloid fibrils, illustrating their value as structural probes to study the molecular mechanisms of
amyloid fibril formation. Combined with their amenability to
protein engineering techniques to improve their stability and solubility, these findings support the suggestion that
nanobodies can potentially be developed as
therapeutics to combat
protein misfolding diseases.