| Summary: | 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 (P<sub>IB</sub>-ATPases) allows flux of heavy metals and are key players in metal detoxification, critical for human health, crops, and survival of pathogens. Nevertheless, P<sub>IB</sub>-ATPases remain poorly understood at a molecular level. In this study, nanobodies (Nbs) are selected against the zinc-transporting P<sub>IB</sub>-ATPase ZntA from <i>Shigella sonnei</i> (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 P<sub>IB</sub>-ATPases.
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