Isolation and structural characterization of a Zn2+-bound single-domain antibody against NorC, a putative multidrug efflux transporter in bacteria

• Published in: The Journal of biological chemistry 2020-01, Vol.295 (1), p.55-68
• Main Authors: Kumar, Sushant, Mahendran, Ithayaraja, Athreya, Arunabh, Ranjan, Rakesh, Penmatsa, Aravind
• Format: Article
• Language: English
• Subjects: antimicrobial efflux; camelid antibody; drug resistance; efflux pump; isothermal titration calorimetry (ITC); major facilitator superfamily (MFS); MRSA diagnosis; multidrug transporter; NorC; Protein Structure and Folding; single-domain antibody (sdAb, nanobody); X-ray crystallography; zinc
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1074/jbc.RA119.010902

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 Escherichia 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 strains that express efflux transporters such as NorC in hospital and community settings.