Nanobody development for therapeutically targeting Vacuolar H+-ATPases

Abstract

The vacuolar H+-ATPase (V-ATPase, V1Vo) is a dedicated proton pump that is highly conserved amongst eukaryotes, and is necessary for pH homeostasis within subcellular compartments. The V-ATPase consists of two subcomplexes: the soluble V1 responsible for hydrolyzing ATP, and the membrane integral Vo responsible for proton translocation across membranes. V1 and Vo are each comprised of multiple subunits, A3B3CDE3FG3 and ac8c'c"def Voa1 respectively. Many basic cellular functions depend on the differential pH gradient across cellular membranes to operate properly, making regulation of V-ATPases through "reversible disassembly" immensely important. Global loss of V-ATPase activity is lethal to all mammalian cell types, while aberrant activity and incorrectly localized V-ATPase results in various disease states. Current therapeutics struggle to target specific V-ATPase populations, and as a potential solution to this problem we generated 94 nanobody clones against the yeast nanodisc reconstituted Vo (VoND). Nanobodies (Nbs) are the small 15 kDa VHH domain isolated from heavy-chain only antibodies that are known for their high specificity. In this dissertation we describe the characterization of three α-yeast VoND Nbs, N27, N125, and N2149. Using an ATPase assay, we found that N27, but not N125 or N21149, fully inhibited the activity of assembled V-ATPase. Contrastingly, N2149, but not N27 or N125, was found to inhibit the assembly of the two subcomplexes. BLI was used to identify the binding affinity of each Nb, with affinities being observed in the nM-pM range. High-resolution structures obtained from cryoEM revealed the subunit specificity of each Nb, with N27 and N125 found to bind the c-ring in different stoichiometries, and N2149 found to bind the d subunit. Furthermore, we determined that N125 has cross affinity for the human enzyme. Overall, this study provides evidence that novel nanobody mediated inhibition of assembly or activity of V-ATPases is an effective technique with broader implications of nanobody development into therapeutics.