EMD-48311

Single-particle
3.7 Å
EMD-48311 Deposition: 13/12/2024
Map released: 05/02/2025
Last modified: 05/02/2025
Overview 3D View Sample Experiment Validation Volume Browser Additional data Links
Overview 3D View Sample Experiment Validation Volume Browser Additional data Links

EMD-48311

Yeast V-ATPase Vo proton channel bound to nanobody 2WVA149

EMD-48311

Single-particle
3.7 Å
EMD-48311 Deposition: 13/12/2024
Map released: 05/02/2025
Last modified: 05/02/2025
Overview 3D View Sample Experiment Validation Volume Browser Additional data Links
Sample Organism: Saccharomyces cerevisiae, Lama glama
Sample: Yeast V-ATPase Vo proton channel subcomplex bound to Nanobody 2WVA149
Fitted models: 9mj4 (Avg. Q-score: 0.455)

Deposition Authors: Wilkens S , Knight K
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
(2025) bioRxiv
PUBMED: 39829782
DOI: doi:10.1101/2025.01.10.632502
ISSN: 2692-8205
Abstract:
The vacuolar ATPase (V-ATPase; V1Vo) 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 Vo proton channel subcomplex. Here, we present an in-depth characterization of three anti-Vo Nbs using biochemical and biophysical in vitro experiments. We find that the Nbs bind Vo 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 Vo on the lumen side of the complex. Additionally, we show that one of the Nbs raised against yeast Vo can pull down human V-ATPase (HsV1Vo). Our research demonstrates Nb versatility to target and modulate the activity of the V-ATPase, and highlights the potential for future therapeutic Nb development.