9mj4
Yeast V-ATPase Vo proton channel bound to nanobody 2WVA149
Structural highlights
FunctionVATL2_YEAST Proton-conducting pore forming subunit of the membrane integral V0 complex of vacuolar ATPase. V-ATPase is responsible for acidifying a variety of intracellular compartments in eukaryotic cells.[1] [2] Publication Abstract from PubMedThe vacuolar ATPase (V-ATPase; V(1)V(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(o) proton channel subcomplex. Here, we present an in-depth characterization of three anti-V(o) Nbs using biochemical and biophysical in vitro experiments. We find that the Nbs bind V(o) with high affinity, with one Nb inhibiting holoenzyme activity and another one preventing enzyme assembly. Using cryoEM, we find that two of the Nbs bind the c subunit ring of the V(o) on the lumen side of the complex. Additionally, we show that one of the Nbs raised against yeast V(o) can pull down human V-ATPase (HsV(1)V(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. Monoclonal nanobodies alter the activity and assembly of the yeast vacuolar H(+)-ATPase.,Knight K, Park JB, Oot RA, Khan MM, Roh SH, Wilkens S bioRxiv [Preprint]. 2025 Jan 11:2025.01.10.632502. doi: , 10.1101/2025.01.10.632502. PMID:39829782[3] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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