9MJ4

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

Summary for 9MJ4

• Entry DOI: 10.2210/pdb9mj4/pdb
• Related: 9e76 9e7l
• EMDB information: 47659 47679 48311
• Descriptor: V-type proton ATPase subunit c', V-type proton ATPase subunit c'', V0 assembly protein 1, ... (9 entities in total)
• Functional Keywords: vacuolar atpase, vo proton channel, lipid nanodisc, nanobody, proton transport
• Biological source: Lama glama; More
• Total number of polymer chains: 16
• Total formula weight: 368901.93

Authors

Wilkens, S.,Knight, K. (deposition date: 2024-12-13, release date: 2025-02-05, Last modification date: 2025-05-14)

Primary citation

Knight, K.,Park, J.B.,Oot, R.A.,Khan, M.M.,Roh, S.H.,Wilkens, S.
Monoclonal nanobodies alter the activity and assembly of the yeast vacuolar H + -ATPase.
Biorxiv, 2025

PubMed Abstract: The vacuolar ATPase (V-ATPase; VV) 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 proton channel subcomplex. Here, we present an in-depth characterization of three anti-V Nbs using biochemical and biophysical experiments. We find that the Nbs bind V 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 subunit ring of the V on the lumen side of the complex. Additionally, we show that one of the Nbs raised against yeast V can pull down human V-ATPase (VV). Our research demonstrates Nb versatility to target and modulate the activity of the V-ATPase, and highlights the potential for future therapeutic Nb development.

PubMed: 39829782
DOI: 10.1101/2025.01.10.632502

Experimental method

ELECTRON MICROSCOPY (3.7 Å)