Development of nanobodies as theranostic agents against CMY-2-like class C β-lactamases

Cawez, F.; Mercuri, P. S.; Yanez, F. M.; Maalouf, R.; Vandevenne, M.; Kerff, F.; Guerin, V.; Mainil, J. G.; Thiry, D.; Salmon, M.; Vanderplasschen, A.; Lafaye, P.; Ayme, G.; Bogaerts, P.; Dumoulin, M.; Galleni, M.

doi:10.1101/2022.07.01.498528

bioRxiv · 10.1101/2022.07.01.498528

Development of nanobodies as theranostic agents against CMY-2-like class C β-lactamases

Cawez, F.·Mercuri, P. S.·Yanez, F. M.·Maalouf, R.·Vandevenne, M.·Kerff, F.·Guerin, V.·Mainil, J. G.·Thiry, D.·Salmon, M.·Vanderplasschen, A.·Lafaye, P.·Ayme, G.·Bogaerts, P.·Dumoulin, M.·Galleni, M.

View original source ↗Export metadata ↓

Abstract

Soluble single-domain fragments derived from the unique variable region of camelid heavy-chain antibodies (VHHs) against enzymes may behave as potent inhibitors. The immunization of alpacas with the CMY-2 {beta}-lactamase led to the isolation of three VHHs that specifically recognized and inhibited CMY-2. The structure of the complex VHH cAbCMY-2(254)/CMY-2 was determined by X-ray crystallography. We showed that the epitope is close to the active site and that the CDR3 of the VHH protrudes in the catalytic site. The {beta}-lactamase inhibition was found to follow a mixed profile with a predominant non-competitive component. The three isolated VHHs recognized overlapping epitopes since they behaved as competitive binder. Our study identified a binding site that can be targeted by a new class of {beta}-lactamases inhibitors designed with the help of a peptidomimetic approach. Furthermore, the use of mono or bivalent VHH and rabbit polyclonal anti-CMY-2 antibodies enable the development of the first generation of ELISA test for the detection of CMY-2 produced by resistant bacteria. IMPORTANCEThe still increasing antimicrobial resistance in human clinic or veterinary medicine is a major threat for modern chemotherapy. Beside the major caution in the use of current antibiotics, it is important to develop new classes of antibiotics. This work was focused on {beta}-lactamases that are the enzymes involved in the hydrolysis of the major class of antibiotics, the {beta}-lactam compounds. We selected camelid antibodies that inhibit CMY-2, a class C {beta}-lactamase produced by bacteria isolated from the veterinary and human settings. We characterized the conformational epitope present in CMY-2 in order to create a new family of inhibitors based on the paratope of the antibody. Finally, we designed a primary version of a detection system based on an ELISA using VHH and polyclonal antibodies.

Explore related subjects

biochemistry Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Cawez, F., Mercuri, P. S., Yanez, F. M., Maalouf, R., Vandevenne, M., Kerff, F., Guerin, V., Mainil, J. G., Thiry, D., Salmon, M., Vanderplasschen, A., Lafaye, P., Ayme, G., Bogaerts, P., Dumoulin, M., Galleni, M.. 2022-07-03. Development of nanobodies as theranostic agents against CMY-2-like class C β-lactamases. https://doi.org/10.1101/2022.07.01.498528

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

EngA adopts multiple distinct conformational states during ribosome assembly

Ribosome assembly, a highly coordinated and crucial cellular process, is assisted by various assembly factors, including GTPases. EngA, an essential P-loop GTPase, is involved in late-stage assembly of the 50S ribosomal subunit. The structures of 50S ribosome in complex with EngA-bound GMPPNP, and EngA-bound GDP alone, without 50S subunit, have been reported previously. However, the molecular mechanism of EngA GTP hydrolysis-assisted 50S maturation remains poorly understood, and structures of the 50S ribosome in complex with EngA-bound GDP have yet to be deciphered. We have reported cryo- EM structures of the Mycobacterium smegmatis 50S ribosome in complex with EngA-bound GTP{gamma}S or GDP at 2.8 - 3.5 [A] resolution. Structural analysis revealed that EngA and 50S subunit undergo a coordinated structural rearrangement, and an assembly intermediate is formed in which the stem-loop of H80 makes base-pair interactions with the junction of H89 and H90, forming a unique stem-loop-junction (SLJ) motif. EngA-bound GDP adopts distinctly different conformations in its ribosome-bound and unbound states. In summary, this study proposed a multi-state conformational model for EngA-assisted ribosome assembly.

biochemistry↗

bioRxiv2026

Modular assembly of mitochondrial complex III from a dynamic matrix domain pool

Mitochondrial complex III is a central component of the cellular energy metabolism. It is an obligate dimer of catalytic and accessory subunits that are encoded by two genomes. How its large matrix domain, formed by the core proteins Qcr1 and Qcr2, is built and joined to the membrane part and during assembly is not known. Here we show that Qcr1 and Qcr2 preassemble independently of complex III into matrix domain modules. Cryo-EM structures from a cardiolipin-deficient yeast strain and of recombinant protein resolved single, double and triple matrix domains, which are linked by the C-terminal tail of Qcr1. In the mature complex III, the same tail binds cytochrome b and cardiolipin. The modules form a dynamic pool, in which assembly species interconvert and exchange subunits. Mutations at the module interfaces, including mimics of human disease variants, reshape this pool and deletion of the Qcr1 tail impairs respiratory growth. Substitution of a tyrosine residue linked to parkinsonism exposes a conserved cysteine residue to oxidation. A cryo-EM structure of an assembly intermediate shows a cap of two matrix domains bound to a monomeric membrane module of cytochrome b, Qcr7, Qcr8 and cytochrome c1. We propose that complex III receives its matrix domain from a dynamic pool of prebuilt modules, which organizes the dimerization of the complex.

biochemistry↗

bioRxiv2026

Vasopressin V2 receptor phosphopeptide reshapes the conformational landscape of arrestin-2 beyond C-tail detachment

Binding of phosphorylated GPCR tails influences arrestin conformation, but the contribution of this interaction to the interdomain rotation associated with activation remains incompletely characterised. In particular, it remains unclear whether displacement of the arrestin C-tail is sufficient to favor larger interdomain rotations or whether the bound phosphotail exerts an additional effect on the conformational landscape. Here, we used enhanced-sampling simulations to characterise the conformational free-energy landscapes of basal (unbound) and V2Rpp-bound arrestin-2. We find that V2Rpp binding broadened the range of energetically accessible interdomain rotation angles relative to basal arrestin-2. However, the pronounced rotation observed in the experimental structures, while accessible, was not strongly favored. Strikingly, C-tail displacement alone does not reproduce this effect, as arrestin-2 retains a strong preference for small interdomain rotation angles in the absence of V2Rpp. Moreover, the C-tail-detached state is energetically unfavorable in the absence of V2Rpp. Together, these findings quantify the effects of V2Rpp binding on the interdomain rotation of arrestin-2 and disentangle the effects of phosphopeptide binding from those of C-tail detachment.

biochemistry↗