Monoclonal nanobodies alter the activity and assembly of the yeast vacuolar H⁺-ATPase

The vacuolar ATPase (V-ATPase; V₁V_o) is a multi-subunit rotary nanomotor proton pump that acidifies organelles in virtually all eukaryotic cells, and extracellular spaces in some specialized tissues of higher organisms. Evidence suggests that metastatic breast cancers mislocalize V-ATPase to the plasma membrane to promote cell survival and facilitate metastasis, making the V-ATPase a potential drug target. We have generated a library of camelid single-domain antibodies (Nanobodies; Nbs) against lipid-nanodisc reconstituted yeast V-ATPase V_oproton channel subcomplex. Here, we present an in-depth characterization of three anti-V_oNbs using biochemical and biophysicalin vitroexperiments. We find that the Nbs bind V_owith high affinity, with one Nb inhibiting holoenzyme activity and another one preventing enzyme assembly. Using cryoEM, we find that two of the Nbs bind thecsubunit ring of the V_oon the lumen side of the complex. Additionally, we show that one of the Nbs raised against yeast V_ocan pull down human V-ATPase (HsV₁V_o). Our research demonstrates Nb versatility to target and modulate the activity of the V-ATPase, and highlights the potential for future therapeutic Nb development.