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

Kumar, S and Mahendran, I and Athreya, A and Ranjan, R and Penmatsa, A (2020) Isolation and structural characterization of a Zn2+-bound single-domain antibody against NorC, a putative multidrug efflux transporter in bacteria. In: The Journal of biological chemistry, 295 (1). pp. 55-68.

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Abstract

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 Ã 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.

Item Type:	Journal Article
Publication:	The Journal of biological chemistry
Publisher:	NLM (Medline)
Additional Information:	The Copyright of this article belongs to the Author(s).
Keywords:	HEAVY-CHAIN ANTIBODIES; MOLECULAR-DYNAMICS; CRYSTAL-STRUCTURE; NANOBODIES; SEQUENCE; TOOLS; MECHANISMS; PUMPS
Department/Centre:	Division of Biological Sciences > Molecular Biophysics Unit
Date Deposited:	23 Jan 2020 09:18
Last Modified:	08 Dec 2022 06:19
URI:	https://eprints.iisc.ac.in/id/eprint/64358

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