Isolation and Characterization of Nanobodies against a Zinc-Transporting P-Type ATPase

Abstract

P-type ATPases form a large and ubiquitous superfamily of ion and lipid transporters that use ATP (adenosine triphosphate) to carry out their function. The IB subclass (PIB-ATPases) allows flux of heavy metals and are key players in metal detoxification, critical for human health, crops, and survival of pathogens. Nevertheless, PIB-ATPases remain poorly understood at a molecular level. In this study, nanobodies (Nbs) are selected against the zinc-transporting PIB-ATPase ZntA from Shigella sonnei (SsZntA), aiming at developing tools to assist the characterization of the structure and function of this class of transporters. We identify six different Nbs that bind detergent stabilized SsZntA. We further assess the effect of the Nbs on the catalytic function of SsZntA, and find that five nanobodies associate without affecting the function, while one nanobody significantly reduces the ATPase activity. This study paves the way for more refined mechanistical and structural studies of zinc-transporting PIB-ATPases.

Therapeutic Nanobodies Targeting Cell Plasma Membrane Transport Proteins: A High-Risk/High-Gain Endeavor

“…Targeting and modulating pathogen-derived P-type ATPases is a promising strategy for the development of new antibiotics, antifungals, vaccines and herbicides, as these ATPases play important roles in the survival of pathogens ( Table 2 ) [ 113 ]. The immunization of a llama with purified SsZntA and panning on biotinylated SsZntA resulted in the identification of multiple SsZnt-targeting nanobodies that could be grouped into three families [ 114 ]. One of the retrieved SsZntA-targeting nanobodies could selectively target the ZntA ATPase and significantly reduced its pump function by up to 50% [ 114 ].…”

“…The immunization of a llama with purified SsZntA and panning on biotinylated SsZntA resulted in the identification of multiple SsZnt-targeting nanobodies that could be grouped into three families [ 114 ]. One of the retrieved SsZntA-targeting nanobodies could selectively target the ZntA ATPase and significantly reduced its pump function by up to 50% [ 114 ]. However, the mechanism-of-action of this inhibition is not yet fully understood [ 114 ].…”

“…One of the retrieved SsZntA-targeting nanobodies could selectively target the ZntA ATPase and significantly reduced its pump function by up to 50% [ 114 ]. However, the mechanism-of-action of this inhibition is not yet fully understood [ 114 ].…”

Genome-wide Identification and Function Analysis of HMAD Gene Family in Cotton (Gossypium Spp.)

“…PIB-type ATPase lpg1024 (LpCopA) from L. pneumophila demonstrated that Cu 2+ ion-entry path involves two ion-binding sites: one transient Met148-Cys382 site and one intramembranous site formed by trigonal coordination to Cys384, Asn689, and Met717 [27]. One nanobodies (Nbs) selected against the zinc-transporting PIB-2-ATPases ZntA from Shigella sonnei (SsZntA), signi cantly reduces the ATPase activity [28]. The multifunctional P1B-4-ATPase CzcP is part of the cobalt, zinc, and cadmium resistance system from the metal-tolerant, model organism Cupriavidus metallidurans, because of an evolutionarily adapted exibility in the TM region likely afforded CzcP the ability to transport Cd 2+ and Zn 2+ in addition to Co 2+ [29].…”

Special Issue: Nanobody

“…Further exploiting their ability of bind to cavities and active sites of the target protein, with Nb9, the authors identified a highly selective inhibitor of the ATPase activity of SsZntA. These nanobodies provide a versatile toolset for structural and functional studies of this subset of ATPases [7]. Focusing on more therapeutic application, nanobodies can be a rich source of neutralizing anti-viral reagents.…”