MgATP hydrolysis destabilizes the interaction between subunit H and yeast V1-ATPase, highlighting H's role in V-ATPase regulation by reversible disassembly

Abstract

Vacuolar H-ATPases (V-ATPases; VV-ATPases) are rotary-motor proton pumps that acidify intracellular compartments and, in some tissues, the extracellular space. V-ATPase is regulated by reversible disassembly into autoinhibited V-ATPase and V proton channel sectors. An important player in V-ATPase regulation is subunit H, which binds at the interface of V and V H is required for MgATPase activity in holo-V-ATPase but also for stabilizing the MgADP-inhibited state in membrane-detached V However, how H fulfills these two functions is poorly understood. To characterize the H-V interaction and its role in reversible disassembly, we determined binding affinities of full-length H and its N-terminal domain (H) for an isolated heterodimer of subunits E and G (EG), the N-terminal domain of subunit (), and V lacking subunit H (VΔH). Using isothermal titration calorimetry (ITC) and biolayer interferometry (BLI), we show that H binds EG with moderate affinity, that full-length H binds weakly, and that both H and H bind VΔH with high affinity. We also found that only one molecule of H binds VΔH with high affinity, suggesting conformational asymmetry of the three EG heterodimers in VΔH. Moreover, MgATP hydrolysis-driven conformational changes in V destabilized the interaction of H or H with VΔH, suggesting an interplay between MgADP inhibition and subunit H. Our observation that H binding is affected by MgATP hydrolysis in V points to H's role in the mechanism of reversible disassembly.

Role of the H subunit C-terminal domain in the assembly of the vacuolar H+-ATPase

“…We have recently observed that transient MgATP hydrolysis on V1Hwt, which, unlike wild type V1, is not in the MgADP inhibited state (Fig. 2B), lowers H's affinity for V1, and we reasoned Role of subunit H in V-ATPase assembly that this destabilization of the V1-H interaction is caused by MgATP hydrolysis driven conformational changes at the catalytic sites and the central (DF) and peripheral stalks (EG1-3) (24). We propose that an allosteric structural change at the open catalytic site driven by release of inhibitory MgADP from the closed catalytic site in autoinhibited, wild type V1 (by a yet unknown mechanism) leads to the detachment of HCT from its inhibitory position at the open site, making it in turn available to bind aNT on Vo.…”

“…Reversible disassembly requires the concerted breakage and reforming of subunit-subunit interactions, most of which involve the peripheral stator subunits E,G,H,C, and aNT. Using purified subunits and their domains, we found that while the head domain of C (Chead) and the N-terminal domain of H (HNT) bind EG with nanomolar affinity, C's foot domain (Cfoot), EG and H, all bind aNT weakly (23,24). It was therefore suggested that the V1-Vo interface is constituted by multiple low-affinity interactions that result in high-avidity binding.…”

“…2B,C, blue trace and bar). Although MgATPase activity is measured in an ATP regenerating system, the activity of V1ΔH decreases over time as MgADP gets trapped in a closed catalytic site, leading to the MgADP inhibited state (18,24). V1Hwt, which distinguishes itself from wild type V1 by having only ~0.4 instead of 1.3 mol/mol ADP in catalytic sites (32), exhibits an initial MgATPase activity of ~ 4.95 ± 0.55 U/mg before becoming MgADP inhibited ( Fig.…”

“…Details of individual experiments have been described in the respective figure legends. The affinity of interaction between V1ΔC and MBP-C was measured using anti-mouse IgG Fc capturing biosensors (FortéBio, AMC biosensors catalog number 18-5088) as described (24).…”

Estudo bioquímico comparativo da (Na+ , K+)-ATPase branquial de Macrobrachium amazonicum (Heller, 1862) habitante de regiões continentais

“…Este mecanismo resulta na interrupção da hidrólise do ATP e do transporte de prótons, sendo geralmente desencadeado em resposta à diminuição dos níveis de glicose Forgac, 2007;Cipriano et al, 2008;Jefferies et al, 2008;Toei et al, 2010). A dissociação dos domínios é importante para economia de energia quando em baixas concentrações de ATP e glicose, uma vez que o domínio V1 isolado não realiza a hidrolise do ATP e o domínio V0 é bloqueado durante o processo para que não ocorra o transporte passivo de prótons (Parra et al, 2000;Diab et al, 2009;Benlekbier et al, 2012;Couoh-Cardel & Wilkens, 2015;Sharma et al, 2018;Hayek et al, 2019). Durante o processo de dissociação, a subunidade C tem um papel fundamental para que o processo ocorra e se separa completamente dos domínios V1 e V0 (Iwata et al, 2004;Jefferies et al, 2008;Tabke et al, 2014).…”