Monoclonal Nanobodies Alter the Activity and Assembly of the Yeast Vacuolar H-ATPase

Overview

Journal bioRxiv

Date 2025 Jan 20

PMID 39829782

Authors

Kassidy Knight

Jun Bae Park

Rebecca A Oot

Md Murad Khan

Soung-Hun Roh

Stephan Wilkens

Affiliations

Soon will be listed here.

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.

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