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Publication Details
AFRICAN RESEARCH NEXUS
SHINING A SPOTLIGHT ON AFRICAN RESEARCH
biochemistry, genetics and molecular biology
Allosteric inhibition of VIM metallo-β-lactamases by a camelid nanobody
Biochemical Journal, Volume 450, No. 3, Year 2013
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Description
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. © The Authors.
Authors & Co-Authors
Sohier, Jean S.
Belgium, Liege
Université de Liège
Laurent, Clémentine
Belgium, Liege
Université de Liège
Chevigné, Andy
Luxembourg, Strassen
Luxembourg Institute of Health
Pardon, Els
Belgium, Ghent
Vlaams Instituut Voor Biotechnologie
Belgium, Brussels
Vrije Universiteit Brussel
Srinivasan, Vasundara
Belgium, Ghent
Vlaams Instituut Voor Biotechnologie
Belgium, Brussels
Vrije Universiteit Brussel
Wernery, Ullrich
United Arab Emirates, Dubai
Central Veterinary Research Laboratory Dubai
Lassaux, Patricia
Belgium, Liege
Université de Liège
Steyaert, Jan
Belgium, Ghent
Vlaams Instituut Voor Biotechnologie
Belgium, Brussels
Vrije Universiteit Brussel
Galleni, Moreno
Belgium, Liege
Université de Liège
Statistics
Citations: 28
Authors: 9
Affiliations: 5
Identifiers
Doi:
10.1042/BJ20121305
ISSN:
02646021
e-ISSN:
14708728