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

Stuti Sharma, Rebecca A. Oot, Stephan Wilkens
2018 Journal of Biological Chemistry  
Vacuolar H + -ATPases (V-ATPases; V1Vo-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 V1-ATPase and Vo proton channel sectors. An important player in V-ATPase regulation is subunit H, which binds at the interface of V1 and Vo. H is required for MgATPase activity in holo V-ATPase, but also for stabilizing the MgADP inhibited state in membrane detached
more » ... membrane detached V1. However, how H fulfills these two functions is poorly understood. To characterize the H-V1 interaction and its role in reversible disassembly, we determined binding affinities of full length H and its N-terminal domain (HNT) for an isolated heterodimer of subunits E and G (EG), the N-terminal domain of subunit a (aNT), and V1 lacking subunit H (V1ΔH). Using isothermal titration calorimetry (ITC) and biolayer interferometry (BLI), we show that HNT binds EG with moderate affinity, that full length H binds aNT weakly, and that both H and HNT bind V1ΔH with high affinity. We also found that only one molecule of HNT binds V1ΔH with high affinity, suggesting conformational asymmetry of the three EG heterodimers in V1ΔH. Moreover, MgATP hydrolysis-driven conformational changes in V1 destabilized the interaction of H, or HNT, with V1ΔH, suggesting an interplay between MgADP inhibition and subunit H. Our observation that H binding is affected by MgATP hydrolysis in V1 points to H's role in the mechanism of reversible disassembly. ________________________________________ Vacuolar ATPases (V-ATPases; V1Vo-ATPases) are ATP dependent proton pumps present in all eukaryotic cells. Typically, the V-ATPase is localized on the endomembrane system, where the enzyme acidifies intracellular compartments, a process essential for pH homeostasis, protein trafficking, endocytosis, hormone secretion, mTOR signaling and lysosomal degradation (1). The V-ATPase is also present on the plasma membrane of certain specialized cells such as osteoclasts, renal cells, the vas deferens and the epididymis where the enzyme acidifies the extracellular environment. V-ATPase's proton pumping activity has been linked to numerous human diseases including osteoporosis and -petrosis (2,3), renal tubular a c i d o s i s ( 4 ) , m a l e i n f e r t i l i t y ( 5 ) , neurodegeneration (6), diabetes (7), viral infection Subunit H Interactions at the V1-Vo interface 1, and cancer (9), making the enzyme a valuable drug target (10,11). The V-ATPase shares a similar architecture and catalytic mechanism with the F-ATP synthase such that it consists of a water soluble ATP hydrolyzing machine (V1) and a membrane integral proton channel (Vo), which are structurally and functionally coupled via a central and multiple peripheral stalks (12) (13) (14) . The subunit composition of the V-ATPase from yeast is A3B3CDE3FG3H for the cytosolic V1 (15) and ac8c'c"def for the membrane integral Vo (16,17). The subunit architecture of the V-ATPase has been studied by electron microscopy (EM) and s ev er al lo w to in ter med iate r es o lu tio n reconstructions of bovine, yeast and insect V-ATPase are available, which, together with X-ray crystal structures of individual subunits and subcomplexes of yeast V-ATPase and bacterial homologs, have provided a detailed model of the subunit architecture of the eukaryotic V-ATPase complex (17-22) (Fig. 1A) . V-ATPase is a rotary motor enzyme (14) . ATP hydrolysis in the catalytic A3B3 hexamer is coupled to rotation of the proteolipid ring (c8c'c") via a central rotor made of subunits D, F and d, with concurrent proton translocation at the interface of the proteolipid ring and the C-terminal domain of subunit a (aCT). During rotary catalysis, the motor is stabilized by a peripheral stator complex consisting of three peripheral stalks constituted by heterodimers of subunits E and G (hereafter referred to as EG1-3) that connect the A3B3 hexamer to the N-terminal domain of the membrane bound a subunit (aNT) via the single copy 'collar ' subunits H and C (19,21) (Fig. 1A) . Three intermediates (referred to as rotational states 1-3), in which the central rotor is spaced 120º relative to the catalytic hexamer and subunit a, have been visualized in the yeast enzyme by cryoEM (21). Unlike the related F-ATP synthase, eukaryotic V-ATPase is regulated by a unique mechanism referred to as reversible disassembly wherein upon receiving cellular signals, V1 dissociates from Vo and the activity of both sectors is silenced (22-26) (Fig. 1B) . Reversible disassembly was first observed in yeast (27) and insects (28), but the process has recently also been observed in higher animals, including human (29-32). Studies in yeast have shown that during enzyme disassembly, subunit C is released into the cytosol by an unknown mechanism and reincorporated during reassembly (27). Owing to regulation of the V-ATPase by reversible disassembly, the peripheral stator subunit interactions at the V1-Vo interface draw particular attention as they must be sufficiently strong to withstand the torque generated during ATP hydrolysis, but at the same time, they must be vulnerable enough to allow breaking on a timescale for reversible disassembly to occur efficiently. We previously characterized the interaction of the collar subunit C with EG and aNT, and found that while the head domain of C (Chead) binds EG with high affinity (Chead-EG3; see Fig. 1A ), its foot domain (Cfoot), as well as EG, both interact weakly with aNT, resulting in a high-avidity ternary interface (EG2-aNT-Cfoot) in holo V-ATPase (33,34). Another collar subunit at the V1-Vo interface is subunit H, and while deletion of C prevents stable assembly of V1 and Vo (35), deletion of H results in the formation of a V1Vo (ΔH) complex that lacks MgATPase and proton pumping activities (36,37). Moreover, while C is released into the cytosol upon disassembly of V1Vo, H remains stably associated with V1 ( Fig. 1B) . The crystal structure of H revealed that it consists of a larger N-terminal (HNT) and smaller C-terminal domain (HCT) connected by a short linker (38). Previous functional characterization of HNT and HCT suggested that while HNT is required for MgATPase activity in V1Vo , HCT has a dual function in that it is required for both coupling of V1's ATPase activity to proton pumping in V1Vo (37), and for inhibition of MgATPase activity in membrane detached V1 (39). The dual role and functional separation of HNT and HCT along with differences in regulatory function compared to C is not well understood and prompted the analyses of the interactions of Subunit H Interactions at the V1-Vo interface 2 by guest on July 21, 2018 Downloaded from H, HNT and HCT with its binding partners in V1 and Vo. We therefore employed recombinant H, HNT and HCT for quantification of their interactions with purified EG, aNT and V1 lacking subunit H (V1ΔH) using isothermal titration calorimetry (ITC) and biolayer interferometry (BLI). We find that HNT binds no more than one of the three EGs on V1ΔH, and that the affinity of this interaction is ~ 40 fold higher than that between HNT and isolated EG, suggesting that HNT prefers a particular conformation of EG on V1. We further found that full length H interacts with V1ΔH with a ~ 70 fold higher affinity than HNT, indicative of a significant contribution of HCT to the binding energy. Furthermore, we show that MgATP hydrolysis driven conformational changes in the catalytic A/B pairs, the central rotor (DF) and the peripheral stalks (EG) destabilize the V1-H interaction until inhibitory MgADP is trapped in a catalytic site. The findings are discussed in context of the mechanism of V-ATPase regulation by reversible disassembly. Results Expression, purification and biophysical characterization of H, HNT, HCT, and aNT(1-372) To understand the role of the V1-Vo interface in the mechanism of reversible disassembly, our lab has previously characterized the interactions between Chead, Cfoot, EG and aNT (33,34). Interactions involving subunit H, however, are yet to be quantified. Pulldown and yeast two hybrid assays have shown that H is able to bind the Nterminal region of subunit E (40). In addition, EM and crystal structures of V1Vo and V1 respectively, show HNT bound to one of the three EG peripheral stalks (EG1; see Fig. 2A ,B), whereas HCT is seen to either rest on the coiled-coil middle domain of aNT (in V1Vo; Fig. 2A ), or at the bottom of the A3B3 hexamer (in autoinhibited V1) ( Fig. 2B ) (21, 22) . To analyze the interactions of H within the enzyme in more detail, we have expressed H, HNT and HCT separately, and quantified their interactions with recombinant EG, aNT, and V1ΔH purified from yeast. Full length H, HNT (residues 1-354), HCT (residues 352-478), and aNT (residues 1-372) were expressed in E. coli as N-terminal fusions with maltose binding protein (MBP). After amylose affinity capture, fusions were cleaved, and MBP was removed by ion exchange and size exclusion chromatography, resulting in purified subunits and subunit domains (Fig. 3A) . All proteins eluted near their expected molecular masses on size exclusion chromatography except aNT(1-372), which exists in a dimer-monomer equilibrium as previously described (26,34) (Fig. 3B,C) . Consistent with available structural information, circular dichroism (CD) spectroscopy revealed a high degree of α helical secondary structure, suggesting proper folding of the recombinant polypeptides (Fig. 3D) . Interaction of HNT with EG We previously established that binding of C (or Chead) to isolated EG occurs with high affinity, and that the interaction greatly stabilizes EG (33). To further characterize the interactions at the V1-Vo interface, we set out to determine the affinity of the HNT -EG interaction using ITC. Titration of HNT into EG was exothermic and the binding curve was fit to a single binding site model, revealing a Kd of the interaction of 187 nM. The binding enthalpy (ΔH) and entropy (ΔS) were -8 kcal/mol and 2.5 cal/(mol·K), respectively, with a concomitant free energy change (ΔG) of ~-36 kJ/ mol (Fig. 4A) . Consistent with the ITC titration, size exclusion chromatography of a mixture of EG and an excess of HNT resulted in the formation of a ternary HNT-EG complex (Fig. 4B,C) , and taken together, the data show that HNT forms a stable complex with the EG heterodimer. Previously, we found that the EG's N-terminal right-handed coiled-coil is thermally labile, with a Tm of ~25 ºC (Fig. 4D , blue trace) (29), and that the Tm of EG is increased by about 10 ºC upon complex formation with C(head) (33). To test whether HNT binding has a similar stabilizing effect on EG, thermal unfolding of the individual proteins and EGHNT complex was monitored by recording the CD signal at 222 nm as a function Subunit H Interactions at the V1-Vo interface 3 by guest on July 21, 2018 Downloaded from of temperature. The data show that isolated HNT unfolds with an apparent Tm of ~ 63 ºC (Fig. 4D , green trace). The thermal unfolding curve of the EGHNT complex showed two transitions, one at 25 and one at 64 ºC, suggesting that the stability of EG is not increased upon HNT binding (Fig. 4D black trace). Moreover, as also shown previously (33), isolated EG heterodimer dissociates during native agarose gel electrophoresis, whereas in presence of Chead, the three proteins migrate as a heterotrimeric complex in the electric field. However, consistent with the thermal unfolding data, a complex of EGHNT did not co-migrate on the native gel, but ran as three separate species (Fig. 4E) . Therefore, while both Chead and HNT form a stable complex with EG, the nature of the two interactions are strikingly different. Interaction of H with aNT Prior work from our laboratory has shown that the EG2-aNT-Cfoot junction at the V1-Vo interface (Fig. 1A) is formed by multiple low affinity interactions, and we reasoned that the sum of these interactions provides a high-avidity binding site between V1 and Vo that could be targeted for regulated disassembly (34). Another interaction that is seen in EM reconstructions of the intact V-ATPase, and that must be broken and reformed during reversible disassembly, is between H and aNT (Figs. 1A, 2A) . To estimate the affinity between H and aNT, we performed ITC experiments by titrating H into aNT(1-372) (Fig. 4F) . Subtracting the heat generated from diluting H into buffer from the heat generated from titrating H into aNT(1-372) revealed a weak endothermic binding reaction between the two proteins. Fitting the data to a one site binding model revealed a Kd of 135 µM and ΔH, ΔS and ΔG values of 4 kcal/mol, 36.1 cal/(mol·K) and -26 kJ/mol, respectively. Consistent with our ITC data, a mixture of H and aNT eluted at the same volumes as the individual proteins on size exclusion chromatography (Fig. S1) . The low affinity interaction between H and aNT supports our existing model that V1 binds Vo via several low-affinity interactions that, taken together, result in a high-avidity interface in assembled V1Vo. Interaction of V1ΔH with H, HNT and HCT characterized by BLI Previous experiments showed that H remains bound to V1 even at the low concentrations used in enzyme essays (e.g. ~15 nM) (22, 25, 39) , and under the conditions of electrospray ionization used for native mass spectrometry (15). While the data so far have shown that the affinity of HNT for EG as measured using ITC is moderately high, the observed Kd of ~0.2 µM (Fig. 4A) could not explain above observations, which means that the interaction of H with V1 has to be much stronger (39). We therefore wished to determine the affinity of full-length H as well as HNT and HCT for V1ΔH. The interaction between V1ΔH and MBP tagged H, HNT and HCT was quantified using BLI. MBP tagged proteins were immobilized on anti-mouse capture (AMC) biosensors using an anti-MBP antibody, and the rate of association and dissociation of V1ΔH was measured hereafter. The slow dissociation of MBP tagged H, HNT and HCT from the anti MBP antibodies was subtracted from the V1ΔH dissociation rates for analysis of the kinetic data. BLI experiments for measuring association and dissociation kinetics between V1ΔH and MBP-H/HNT were conducted at five different V1ΔH concentrations, and the resulting association and dissociation curves were fit to a global single site binding model (Fig. 5A,B) . Analysis of the data for MBP-H and MBP-HNT revealed Kds of ∼ 65 pM (Fig. 5A) and ~4.5 nM (Fig. 5B) , respectively. We also tested the binding of V1ΔH to MBP-HCT, but we were not able to determine a Kd as there was no measurable association at low V1ΔH concentrations (<100 nM), with higher V1ΔH concentrations (e.g. 1 µM) resulting in nonspecific binding to the BLI sensors (data not shown). Overall, the interaction of HNT with EG as part of V1ΔH was ~40 fold tighter when compared to the interaction between HNT and isolated EG (as measured using ITC, Fig. 4A) , suggesting that the conformation of EG on V1ΔH is more favorable for HNT binding than the Subunit H Interactions at the V1-Vo interface 4 by guest on July 21, 2018 Downloaded from conformation(s) of isolated EG. In addition, while we could not detect an interaction between HCT and V1ΔH under the conditions of BLI, a ~70 fold higher affinity of V1ΔH for H as compared to HNT suggests a significant contribution of HCT to the V1-H interaction. From our ITC and BLI experiments, we infer that the binding interaction between HNT and EG allows HCT to switch conformations, so that it can either bind aNT (in V1Vo), or subunits B and D (in V1), to efficiently carry out its dual role in reversible disassembly. V1ΔH binds no more than one HNT Since V1ΔH contains three EG heterodimers, we wished to determine whether all three, or only one of the EGs can bind HNT. Purified V1ΔH was mixed with a fivefold molar excess of HNT followed by size exclusion chromatography. Under these conditions, some HNT co-eluted with V1ΔH, with the excess of HNT eluting from the column as a separate, lower molecular weight peak (Fig. 5C,D) . The V1ΔH/HNT complex was concentrated and approximately equal amounts of V1ΔH and V1ΔH/HNT were resolved using SDS-PAGE. The staining of HNT in the purified V1ΔH/ HNT complex was similar to that of single copy subunits in the V1 complex (for example subunit D), indicating that no more than one copy of HNT bound to V1ΔH (Fig. 5E) . Therefore, while there are three EGs in V1ΔH, only one of these is in a conformation that is able to bind HNT with high affinity, highlighting the conformational asymmetry of the peripheral stalks. The interaction of H with V1ΔH is destabilized upon MgATP hydrolysis The preference of HNT for one out of three EGs suggested that the asymmetry of the peripheral stalks originates in the catalytic core (A3B3DF) of V1. Upon MgATP hydrolysis, however, the conformational changes of the catalytic sites from open, to loose, to tight drive counterclockwise rotation of the central rotor along with cyclic structural changes in the peripheral stalks, from EG1 to EG3 to EG2 (41). In addition, based on the structure and nucleotide occupancy of the autoinhibited V1 sector, our lab suggested that HCT inhibits V1-ATPase activity by preferentially binding to an open catalytic site, consequently maintaining inhibitory MgADP in the adjacent closed catalytic site (22). Taken together, HNT's preference for EG1, as well as HCT's role in MgADP inhibition, indicated a potential interplay between the nucleotide occupancy of the catalytic sites and the interaction of V1ΔH with H. To probe the effect of nucleotides on the interaction of H with V1ΔH, we again used BLI. V1ΔH was bound to immobilized MBP-H, and the sensor was then dipped in wells containing buffer, or buffer with 1 mM MgATP, MgADP+Pi or MgAMPPNP. Interestingly, in the presence of MgATP, a biphasic dissociation curve was observed, with an initial dissociation rate that was ~ 6 times faster than the rate in buffer alone (Fig. 6A,B) . However, only ~25% of the bound V1ΔH dissociated with a fast rate, with the remaining 75% coming off the sensor at a rate similar to the dissociation rate in buffer (Fig. 6A,B) . On the other hand, a relatively slower rate of dissociation was observed in the presence of MgADP+Pi and MgAMPPNP. The destabilization of the V1-H interaction upon MgATP hydrolysis came as a surprise to us as the H subunit is known to inhibit V1-ATPase activity (22, 25, 39) . To confirm that the fast dissociation of V1ΔH from MBP-H in wells containing MgATP was specifically due to MgATP binding to V1's catalytic sites, we conducted a similar BLI experiment using V1ΔH treated with N-ethymaleimide (NEM). It is known that NEM modification of a catalytic site cysteine residue prevents binding of nucleotides (42). NEM treated and untreated V1ΔH were bound to MBP-H immobilized on sensors and then dipped in wells containing MgATP, MgADP+Pi or buffer (Fig. 6C,D) . We found that NEM treated V1ΔH no longer showed a fast dissociation rate when dipped in MgATP containing wells, suggesting that MgATP binding to the catalytic sites caused destabilization of the V1-H interaction. However, if the above mentioned fast dissociation rate was a result of only MgATP binding, but not hydrolysis, Subunit H Interactions at the V1-Vo interface 5 by guest on July 21, 2018 Downloaded from we should have observed fast dissociation in the presence of MgAMPPNP, the non-hydrolyzable ATP analog. MgAMPPNP, however, had no effect on the V1ΔH dissociation rate (Fig. 6A , green trace). Taken together, the BLI experiments with NEM modified V1ΔH and in presence of MgAMPPNP suggest that it is MgATP binding and hydrolysis that destabilizes the V1-H interaction. Since both HNT and HCT contribute to the interaction of H with V1ΔH, we also measured the off rate of V1ΔH from immobilized MBP-HNT and found that the HNT/V1ΔH interaction was also destabilized upon MgATP hydrolysis, as seen for H and V1ΔH (Fig. 6E,F) . To verify that V1ΔH bound to H was capable of transient turnover, we purified V1ΔH, incubated it with an excess of H, and resolved the mixture using size exclusion chromatography (Fig. S2) . We found that V1ΔH reconstituted with H (V1ΔH.H) showed ~4.9 ± 0.55 U/mg of MgATPase activity, which was ~30% of the activity of V1ΔH (15.75 ± 1.7 U/mg) (Fig 6G and (22) ). Considering the high affinity interaction between V1ΔH and H with a Kd of ~65 pM, we expected stoichiometric amounts of H in the V1ΔH.H reconstituted complex. However, to exclude the possibility that the observed MgATPase activity in the V1ΔH.H complex was due to sub-stoichiometric binding of H, we performed a pull down experiment, in which a tenfold molar excess of MBP-H bound to amylose resin was used to capture V1ΔH (Fig. S3) . While some MBP-H and V1ΔH appeared in the supernatant and washes of the amylose resin, most of the MBP-H eluted in 10 mM maltose, along with stoichiometrically bound V1ΔH. Elution fraction E1 (Fig. S3A ) exhibited significant MgATPase activity (Fig. S3B) , indicating that a stoichiometric complex of V1ΔH with MBP-H was capable of hydrolyzing MgATP. A consistent feature of the dissociation curve of V1ΔH from H in MgATP was its biphasic nature (Fig. 6A , orange trace) indicating that MgATP hydrolysis dependent destabilization was transient. We found that by using different concentrations of MgATP during dissociation, we were able to regulate the fast phase of the dissociation rate, and consequently the duration of destabilization (Fig. 7) . Not only does this experiment confirm MgATP hydrolysis as being the cause of V1-H destabilization, it also explains why the destabilization is transient. Using Pi release based ATPase assays, it has been shown that MgATPase activity of V1ΔH subsides rapidly with time (25). This rapid decrease in activity, which is also observed in an ATP regenerating assay (Fig. S4) , has been attributed to the trapping of MgADP in a catalytic site, a phenomenon termed MgADP inhibition. We have observed that MgADP inhibition of V1ΔH is more efficient under high Mg 2+ (and by extension high MgATP) concentrations, and that decreasing the initial concentration of MgATP results in delayed MgADP inhibition (Fig. S4 ). In the BLI experiment shown in Fig. 7 , decreasing the concentration of MgATP to 100 µM (Fig. 7 , red trace) and consequently decreasing the rate of MgATP hydrolysis, led to a delay in the culmination of the fast dissociation phase. Taken together, this data suggests that MgATP hydrolysis causes transient destabilization of the V1-H interaction until MgADP inhibition sets in. Discussion V-ATPase is regulated by reversible disassembly, a process that involves the breakage and reformation of several protein-protein interactions at the interface of V1 and Vo. These interactions are mediated by the central rotor of V1 (DF) binding to Vo's subunit d, and the three peripheral stalks (EG1-3) that link the collar subunits H and C to aNT (see Fig. 2A ). Both H and C are two domain proteins, and we previously found that Chead binds EG with high-affinity, whereas Cfoot and EG bind aNT weakly. Here, we analyzed binding of H and HNT to isolated EG, aNT and V1ΔH purified from yeast. We found that the majority of the binding energy between H and V1 is contributed by the interaction between HNT and EG, and that binding of HNT to EG is much stronger when EG is part of V1 compared to isolated EG. However, only one copy of HNT Subunit H Interactions at the V1-Vo interface 6 by guest on July 21, 2018 Downloaded from binds V1ΔH with high-affinity, indicating that the three EGs on V1 are in different conformations, and that only one of these conformations (EG1) is competent for H binding. The three different conformations of the EGs are evident from the crystal structure of auto-inhibited V1 (22) as well as the cryoEM structures of V1Vo (20, 21) . The observation that HNT binds only EG1 as part of V1 with high-affinity suggests that HNT's preference is a result, and not the cause, of the conformational asymmetry of the peripheral stalks, which most likely originates in the catalytic core of V1 (A3B3DF). While we were not able to detect an interaction between isolated HCT and V1ΔH, the observation that intact H binds V1ΔH with significantly higher affinity compared to HNT, suggests that the contact between HCT and A3B3DF seen in the V1 crystal structure (22) contributes to the avidity of the V1-H interaction. In addition, much like the Cfoot-aNT-EG lowaffinity (but high-avidity) ternary junction, we found that H and aNT interact weakly. Taken together, the data support, and extend our earlier findings that V1 and Vo are held together by multiple weak interactions that allow rapid breaking and reforming in response to cellular needs. MgATP hydrolysis dependent destabilization of the V1-H interaction: Studies in yeast have shown that membrane detached V1 has no measurable MgATPase activity, a property of wild type V1 that has been attributed to the presence of the inhibitory H subunit (25). Therefore, our BLI data showing that the V1-H interaction is destabilized in presence of MgATP came as a surprise, as we did not expect V1ΔH.H to be catalytically active. On the other hand, previous biochemical studies had shown that V1ΔH retained ~20% MgATPase activity upon addition of an excess of recombinant H, an observation t h a t , a t t h e t i m e , w a s a t t r i b u t e d t o substoichiometric binding of H (39). However, using pull-down assays, we here show that a stoichiometric complex of V1ΔH with H has indeed transient MgATPase activity, indicating that wild type V1 isolated from yeast is not equivalent to reconstituted V1ΔH.H. One striking difference between V1 and V1ΔH.H is that V1 contains ~1.3 mol/mol of tightly bound ADP, whereas V1ΔH, and by extension V1ΔH.H has only ~0.4 mol/mol ADP (22). This suggests that V1's ATPase activity is inhibited by tightly bound ADP, and that the lack of ADP in V1ΔH.H allows transient MgATP hydrolysis, with the associated conformational changes leading to destabilization of the V1-H interaction on the BLI sensor. MgADP inhibition is a conserved feature of the catalytic headpiece of rotary ATPases, wherein under ATP regenerating conditions, the rate of MgATP hydrolysis decreases due to retention of tightly bound MgADP at a closed catalytic site. The MgADP inhibited state is a conformation offpathway from the catalytic cycle and associated with a structural change in the catalytic site (43). MgADP inhibition has been observed in both the F1-ATPase (e.g. F1 from bovine heart (44)) and Bacillus PS3 (45)), and the cytosolic A1/V1 sector from Thermus thermophilus (46). Parra et al. reported a decrease in MgATPase activity of purified yeast V1ΔH using Pi release assays (25), and we have observed a similar decrease in MgATPase activity of V1ΔH using an ATP regenerating assay system (22) (Fig S4) .
doi:10.1074/jbc.ra118.002951 pmid:29754144 fatcat:jhngfqrs3fhqrboaf3jvf23a6a