Structures of an intact yeast V-ATPase alone and in complex with bacterial effector VopQ
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Abstract
Vacuolar-type ATPase (V-ATPase) is a rotary protein pump involved in proton translocation across various cellular membranes using the energy of ATP hydrolysis. Despite previous studies on bacterial and eukaryotic V-ATPases, information on the intact structure of a eukaryotic V-ATPase is missing. Here we report cryo-EM structures of the intact yeast V-ATPase and this complex bound to a bacterial effector. We reveal the interaction of the elusive regulatory subunit H with its neighboring subunits. Insight for the catalysis mechanism is gained by determining conformations of the catalytic subunits either empty or bound with nucleotides.
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###Reviewer #3:
In this manuscript, Peng et al. report three cryo-EM structures of the yeast V-ATPase holoenzyme, two without VopQ and one bound to the bacterial effector VopQ at 3-3.5A resolution. These structures reveal different functional states of the complex, with the ATPase sites adopting either closed or open conformations, supporting a rotary catalytic mechanism proposed previously. Compared to published structures of V1 or V0 subcomplexes and of the rat holoenzyme, the novelty of the authors' study lies in resolving the regulatory subunit H bound to the yeast holoenzyme at near-atomic resolution. Surprisingly, however, little mechanistic insight is provided by the authors into how this key regulator controls V-ATPase activity. For example, what is the structural explanation for why subunit H is essential for holoenzyme …
###Reviewer #3:
In this manuscript, Peng et al. report three cryo-EM structures of the yeast V-ATPase holoenzyme, two without VopQ and one bound to the bacterial effector VopQ at 3-3.5A resolution. These structures reveal different functional states of the complex, with the ATPase sites adopting either closed or open conformations, supporting a rotary catalytic mechanism proposed previously. Compared to published structures of V1 or V0 subcomplexes and of the rat holoenzyme, the novelty of the authors' study lies in resolving the regulatory subunit H bound to the yeast holoenzyme at near-atomic resolution. Surprisingly, however, little mechanistic insight is provided by the authors into how this key regulator controls V-ATPase activity. For example, what is the structural explanation for why subunit H is essential for holoenzyme activity? How does subunit H inhibit ATP hydrolysis in the V1 subcomplex?
Major comments:
The authors refer to states 1, 2 and 3 throughout their manuscript, without ever introducing these states or explaining the differences. While experts in the V-ATPase and F-ATPase field may be familiar with these states, the manuscript in its current form is not well accessible for non-experts.
It is unclear why the V0V1 sample without VopQ was prepared with AMPNP, but the one with VopQ contained an equimolar mixture of AMPNP and ADP. For better comparison of both structures, it seems it would have been more appropriate to use the same nucleotide conditions. Related to that, the authors state that VopQ locks the holoenzyme in state 2. How can the authors exclude that the addition of ADP caused this effect, especially since VopQ seems substoichiometric (see below)? If VopQ stabilizes state 2, how is this achieved?
The density for VopQ in the authors’ structure is extremely weak, indicating only a subpopulation of particles actually contains VopQ. The authors should try focused classification to better separate VopQ-bound and -free holoenzyme.
Page 6: "Therefore, our data also suggests that subunit H is present in possible disassembled V1 subcomplex and in the holocomplex, ..." It is unclear how the authors' structures or ATPase data allows this conclusion. The authors should explain.
The authors identify specific interaction pairs between subunit H and subunits in V0 and V1. How do mutations at these interfaces affect V-ATPase holoenzyme stability and activity? Mutational analyses would provide an important validation of the structures and insights into the mechanism by which subunit H regulates V-ATPase activity.
The authors mention differences in the stator subunits between the rat and yeast holoenzymes. It would be worthwhile including a figure of this comparison.
The atomic models for the three related cryo-EM structures are poorly refined, with clash scores of >40, ~1.5% Ramachandran outliers and 16-17% rotamer outliers. The proteins and ligands in the various models also have unusually low B-factors for the reported resolutions. The authors must properly refine their atomic coordinates. It is also unclear why three different map sharpening factors are listed for each EM map.
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###Reviewer #2:
In this manuscript, the authors describe cryo-EM structures of the assembled yeast V-ATPase in the presence of the inhibitory nucleotide AMP-PNP and in the presence of VopQ, an inhibitor recently shown to bind to the Vo sector. The structure is reported to be of higher resolution than previous cryo-EM structures of the same yeast enzyme in three rotational states (2015) and the yeast V-ATPase containing the Stv1 isoform (2019), both reported by the Rubinstein lab. As in those structures, there are areas of lower resolution, and the catalytic hexamer shows the highest resolution. Three distinct conformations were observed in the Rubinstein Vph1-V-ATPase cryo-EM structure, potentially corresponding to three rotational states. Here only two states are observed, possibly as a result of the presence of the inhibitory …
###Reviewer #2:
In this manuscript, the authors describe cryo-EM structures of the assembled yeast V-ATPase in the presence of the inhibitory nucleotide AMP-PNP and in the presence of VopQ, an inhibitor recently shown to bind to the Vo sector. The structure is reported to be of higher resolution than previous cryo-EM structures of the same yeast enzyme in three rotational states (2015) and the yeast V-ATPase containing the Stv1 isoform (2019), both reported by the Rubinstein lab. As in those structures, there are areas of lower resolution, and the catalytic hexamer shows the highest resolution. Three distinct conformations were observed in the Rubinstein Vph1-V-ATPase cryo-EM structure, potentially corresponding to three rotational states. Here only two states are observed, possibly as a result of the presence of the inhibitory nucleotide. VopQ inhibition of the intact V-ATPase only occurs in the absence of ATP hydrolysis, and the VopQ-V-ATPase structure, obtained in the presence of AMP-PNP and ADP, appears to enrich the State 2 conformation. However, the VopQ itself is very poorly resolved. Overall, AMP-PNP-bound and VopQ-containing V-ATPase structures do provide some new information, particularly the side-chain interactions with subunit H, but several claims are overstated.
The following issues should be addressed:
The authors do not give sufficient credit to previous work. The statement on lines 50 and 51, "We describe the cryo-EM structures of the first intact eukaryotic holoenzyme V-ATPase complex (V1Vo)..." is simply not true given the previous yeast structures from the Rubinstein lab. The main advance here is in improved resolution (from 6-8 A to 3.1-3.5 A) for two of three rotational states. Overall, the authors need to do a better job of highlighting what is really novel in their study, starting in the Abstract, which does not highlight the new information in the structures here.
The absence of the third rotational state (State 3) is attributed to disassembly of the V-ATPase (lines 64-66). However, this does not make sense given the fact that all three structures were found in the previous studies, and that V-ATPase disassembly is actually inhibited when ATPase activity is inhibited. Instead the absence of this state (which is consistently the least represented) must be associated with either the AMP-PNP inhibition or the number of particles visualized.
From their recent structures showing VopQ binding to the membrane Vo subcomplex, it was expected that VopQ would bind to State 2 of the holoenzyme. Unfortunately, the inhibitor could not be visualized well in the context of the intact enzyme, but there appears to be an enrichment and/or stabilization of State 2 of the V1Vo. However, the VopQ-V-ATPase samples also contain both AMP-PNP and ADP, so the authors should at least discuss whether it is the ADP or the VopQ that led to the stabilization of State 2 (especially given apparent low occupancy of VopQ). This structure did allow more detailed view of the subunit side chain interactions with subunit H than was possible previously. However, the suggestion that this structure was the first demonstration that subunit H was present in the holoenzyme (lines 107-109) is not correct, as this subunit co-purifies with intact V-ATPases and was present in previous structures.
The suggestion in lines 214-217 that this is the "first direct observation of various conformations of subunit pairs in a V-ATPase holoenzyme" is overstated. Conformational changes due to nucleotide binding have been visualized in even higher resolution crystal structures of the conserved bacterial (E. hirae) V1 (ref. 14).
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###Reviewer #1:
Structures are reported of yeast V-ATPase. They are similar to previously reported structures of rat and human V-ATPase, and are consistent with previously established mechanistic models. The major advance is that the new structures include subunit H, which is required for activity of the holoenzyme but inhibits ATPase activity in the isolated V1 component. Unfortunately, the structures do not indicate a mechanistic basis for subunit H activity. Another new feature of the current structures is inclusion of the bacterial effector VopQ, which was previously visualized binding to two sites on the isolated V0 subcomplex. Unfortunately, the density of VopQ in the current structures appears to be extremely poor. In summary, although the visualization of subunit H is an advance, the relative lack of new mechanistic insight from …
###Reviewer #1:
Structures are reported of yeast V-ATPase. They are similar to previously reported structures of rat and human V-ATPase, and are consistent with previously established mechanistic models. The major advance is that the new structures include subunit H, which is required for activity of the holoenzyme but inhibits ATPase activity in the isolated V1 component. Unfortunately, the structures do not indicate a mechanistic basis for subunit H activity. Another new feature of the current structures is inclusion of the bacterial effector VopQ, which was previously visualized binding to two sites on the isolated V0 subcomplex. Unfortunately, the density of VopQ in the current structures appears to be extremely poor. In summary, although the visualization of subunit H is an advance, the relative lack of new mechanistic insight from the current study diminishes my enthusiasm.
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