Paratope Duality and Gullying are Among the Atypical Recognition Mechanisms Used by a Trio of Nanobodies to Differentiate Ebolavirus Nucleoproteins

We had previously shown that three anti–Marburg virus nanobodies (VHH or single-domain antibody [sdAb]) targeted a cryptotope within an alpha-helical assembly at the nucleoprotein (NP) C-terminus that was conserved through half a century of viral evolution. Here, we wished to determine whether an anti–Ebola virus sdAb, that was cross-reactive within the Ebolavirus genus, recognized a similar structural feature upstream of the ebolavirus NP C-terminus. In addition, we sought to determine whether the specificities of a less cross-reactive anti–Zaire ebolavirus sdAb and a totally specific anti–Sudan ebolavirus sdAb were the result of exclusion from this region. Binding and X-ray crystallographic studies revealed that the primary determinant of cross-reactivity did indeed appear to be a preference for the helical feature. Specificity, in the case of the Zaire ebolavirus–specific sdAb, arose from the footprint shifting away from the helices to engage more variable residues. While both sdAbs used CDRs, they also had atypical side-on approaches, with framework 2 helping to accommodate parts of the epitope in sizeable paratope gullies. The Sudan ebolavirus–specific sdAb was more remarkable and appeared to bind two C-terminal domains simultaneously via nonoverlapping epitopes—“paratope duality.” One mode involved paratope gullying, whereas the other involved only CDRs, with CDR3 restructuring to wedge in between opposing walls of an interdomain crevice. The varied routes used by sdAbs to engage antigens discovered here deepen our appreciation of the small scaffold’s architectural versatility and also reveal lucrative opportunities within the ebolavirus NP C-termini that might be leveraged for diagnostics and novel therapeutic targeting.