Isolation and structural characterization of a Zn2+-bound single-domain antibody against NorC, a putative multi-drug efflux transporter in bacteria.
J Biol Chem. 2019 Nov 07;:
Authors: Kumar S, Ithayaraja M, Athreya A, Ranjan R, Penmatsa A
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 14TM helix transporter, NorC, from Staphylococcus aureus We identified this antibody in a yeast display screen built from mononuclear cells isolated from the immunized camel and purified the antibody from E.coli 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 Zn2+-binding site that substitutes for a loop-stabilizing disulfide bond. We performed mutagenesis to compromise the Zn2+-binding site and observed that this change severely hampered antibody stability and its ability to interact with the antigen. The lack of bound Zn2+also made the CDR3 loop highly flexible as observed in all-atom simulations. Using confocal imaging of NorC-expressing E.coli 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 S.aureus (MRSA) strains that express efflux transporters such as NorC in hospital and community settings.
PMID: 31699895 [PubMed - as supplied by publisher]