title: Allosteric inhibition of VIM metallo-β-lactamases by a camelid nanobody.
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.
authors.affiliation: Centre for Protein Engineering, Macromolecules Biologiques Unit, University of Liège, Allée du 6 Août, 13 (B6A), Sart-Tilman, 4000 Liege, Belgium.
authors.forename: Jean S
authors.initials: JS
authors.lastname: Sohier
authors.forename: Clémentine
authors.initials: C
authors.lastname: Laurent
authors.forename: Andy
authors.initials: A
authors.lastname: Chevigné
authors.forename: Els
authors.initials: E
authors.lastname: Pardon
authors.forename: Vasundara
authors.initials: V
authors.lastname: Srinivasan
authors.forename: Ulrich
authors.initials: U
authors.lastname: Wernery
authors.forename: Patricia
authors.initials: P
authors.lastname: Lassaux
authors.forename: Jan
authors.initials: J
authors.lastname: Steyaert
authors.forename: Moreno
authors.initials: M
authors.lastname: Galleni
mesh_terms: D000494:Allosteric Regulation / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D002161:Camelids, New World / Q000276:immunology; D002162:Camelus / Q000276:immunology; D020134:Catalytic Domain; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D061905:Single-Domain Antibodies / Q000494:pharmacology*; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000737:chemistry / Q000276:immunology
chemical_list: D004791:Enzyme Inhibitors; D000939:Epitopes; D061905:Single-Domain Antibodies; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases; C477893:beta-lactamase bla(VIM-4), Pseudomonas aeruginosa