Isolation and structural characterization of a Zn-bound single-domain antibody against NorC, a putative multidrug efflux transporter in bacteria.

Single-chain antibodies from camelids have served as powerful tools ranging from diagnostics and therapeutics to crystallization chaperones meant to study protein structure and function. In this study, we isolated a single-chain antibody from an Indian dromedary camel (ICab) immunized against a bacterial 14 helix transporter, NorC, from We identified this antibody in a yeast display screen built from mononuclear cells isolated from the immunized camel and purified the antibody from after refolding it from inclusion bodies. The X-ray structure of the antibody at 2.15 Å resolution revealed a unique feature within its CDR3 loop, which harbors a Zn-binding site that substitutes for a loop-stabilizing disulfide bond. We performed mutagenesis to compromise the Zn-binding site and observed that this change severely hampered antibody stability and its ability to interact with the antigen. The lack of bound Zn also made the CDR3 loop highly flexible, as observed in all-atom simulations. Using confocal imaging of NorC-expressing spheroplasts, we found that the ICab interacts with the extracellular surface of NorC. This suggests that the ICab could be a valuable tool for detecting methicillin-resistant strains that express efflux transporters such as NorC in hospital and community settings.