Allosteric inhibition of VIM metallo-β-lactamases by a camelid nanobody.

TitleAllosteric inhibition of VIM metallo-β-lactamases by a camelid nanobody.
Publication TypeJournal Article
Year of Publication2013
AuthorsSohier, J. S., C. Laurent, A. Chevigné, E. Pardon, V. Srinivasan, U. Wernery, P. Lassaux, J. Steyaert, and M. Galleni
JournalBiochem J
Volume450
Issue3
Pagination477-86
Date Published2013 Mar 15
ISSN1470-8728
KeywordsAllosteric Regulation, Amino Acid Sequence, Animals, beta-Lactamases, Camelids, New World, Camels, Catalytic Domain, Enzyme Inhibitors, Epitope Mapping, Epitopes, Models, Biological, Models, Molecular, Molecular Sequence Data, Single-Domain Antibodies
Abstract

MβL (metallo-β-lactamase) enzymes are usually produced by multi-resistant Gram-negative bacterial strains and have spread worldwide. An approach on the basis of phage display was used to select single-domain antibody fragments (VHHs, also called nanobodies) that would inhibit the clinically relevant VIM (Verona integron-encoded MβL)-4 MβL. Out of more than 50 selected nanobodies, only the NbVIM_38 nanobody inhibited VIM-4. The paratope, inhibition mechanism and epitope of the NbVIM_38 nanobody were then characterized. An alanine scan of the NbVIM_38 paratope showed that its binding was driven by hydrophobic amino acids. The inhibitory concentration was in the micromolar range for all β-lactams tested. In addition, the inhibition was found to follow a mixed hyperbolic profile with a predominantly uncompetitive component. Moreover, substrate inhibition was recorded only after nanobody binding. These kinetic data are indicative of a binding site that is distant from the active site. This finding was confirmed by epitope mapping analysis that was performed using peptides, and which identified two stretches of amino acids in the L6 loop and at the end of the α2 helix. Because this binding site is distant from the active site and alters both the substrate binding and catalytic properties of VIM-4, this nanobody can be considered as an allosteric inhibitor.

DOI10.1042/BJ20121305
Alternate JournalBiochem. J.
PubMed ID23289540