On 2021-03-25 14:02:08, user Magnus Kjaergaard wrote:
Response to eLife reviewer 3. Our answer in italics.
Reviewer #3 (General assessment and major comments (Required)):
In this manuscript by Hansen et al., the authors describe three low (3.0 to 4.0 Å) resolution crystal structures of Ca2+-ATPase from Listeria, a gram positive bacterium. Two are crystal structures of wild type protein with B eF3- and AlF4- in the absence of Ca2+, thus, likely to represent the E2P ground state and E2~P transition state. The third one is a structure of a G4 mutant, in which 4 Gly residues are inserted into the A-domain -M1 linker, with BeF3- and Ca2+-present in crystallisation, designed to capture the E2P[Ca2+] state. Authors state, however, the three structures are virtually the same and that the E2·BeF3- crystal structure represents a state just prior to ("primed for") dephosphorylation. They also propose that proton counter transport "mechanism" is different from that of SERCA.
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As Listeria Ca2+-ATPase has been studied by a single molecule FRET, its crystal structures will certainly contribute to our understanding of ion pumping. Furthermore, different from SERCA, Listeria Ca2+-ATPase transports only one Ca2+ per ATP hydrolysed. Therefore, how site I is managed is an interesting topic, although lets not forget the same 1:1 stoichiometry is observed with plasma membrane Ca2+-ATPase (PMCA), for which an EM structure appeared in 2018 (ref. 9). The authors indeed find that the Arg795 side chain extends into binding site I. This part is solid and a more elaborate (and interesting) discussion could be made than what is currently described.
Another solid finding is that the two E2·BeF3- crystal structures are similar to the E2·AlF4- crystal structure, although how similar is unclear as a structural superimposition reporting an RMSD is not provided and the presented figure makes it difficult to judge directly; the structures are viewed from almost one direction, which makes it unfeasible to discern the differences in M1 and M2 and in the horizontal rotation of the A-domain. Two or three structures are superimposed, but with cylinders and again viewed from only one direction. As the authors designate that the structures represent H+ occluded states, it is important to clearly show the extracellular gate is really closed to H+ (not only to Ca2+ as well). For completeness, they should also examine the effect of crystal packing on the A-domain position. <br />
A new view of the structures after a 90-degree rotation has been added to Figure S2 and 6 to make it easier to judge domain orientation. Additionally, we have added a new supplementary table S2 containing RMSDs for pairwise alignments of LMCA1 and SERCA structure.<br />
A new supplementary figure S3 has been added, which shows crystal packing of the A domain in the three structures. The packing differs between G4 and WT structures. As the contacts are on the outer surface of the headpiece, we think it is unlikely that they affect any of the structural interpretation in the manuscript, but we have added the following sentence to the discussion of the headpiece orientation: <br />
“The A domain makes different crystal contacts in WT and G4 structures (Figure S3), so changes in the domain orientation should be interpreted with caution. “
With regard to the point that the E2·BeF3- structure is "primed for dephosphorylation", only Fig. 2 (now Figure 3) is shown, in which differences appear to be the path of the TGES loop and the orientation of the Glu167/183 side chain. Their atomic models show that there is a plenty of space for the Glu167 sidechain to take an orientation similar to that of Glu183 in SERCA. The authors should, however, provide an omit annealed Fo-Fc map for the Glu167 side chain and explain why that is the preferred and only orientation. If a Glu side chain is free to move, it could adopt in less than a nanosecond a different orientation. If it does, then the difference in the orientation of the Glu side chain does not sufficiently explain "the rapid dephosphorylation observed in single-molecule studies". The authors place further emphasis on proton occlusion and countertransport. However, this part of the manuscript is more speculative and, as detailed later should, at least, be entirely moved to the Discussion section.
We have added a new supplementary Figure S5 showing an omit annealed Fo-Fc map for Glu167. This shows that the side chain has the preferred location that we discuss. We would like to clarify that the pre-organization of the catalytic side is not merely a question of the rotamer of the side chain of Glu167, but also requires the TGES loop to break interactions to reorganize its backbone structure. This can be seen e.g. in Figure 3C. <br />
Proton occlusion and counter-transport will be addressed below.
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As mentioned, the authors place a larger emphasis on proton countertransport. Here a number of issues show up. First of all, I think they have frequently used the term "occlusion" improperly. From my understanding, occlusion of a site (or ion) means that the site (or ion) is inaccessible from either side of the membrane. This means more than closure of the gates, as the two gates have to stay closed for a substantial length of time (i.e. locked). It is experimentally well established with SERCA that Ca2+ ions are occluded in E1P species. It can be shown that the lumenal gate is closed for Ca2+ in the E2 state. However, that does not necessarily mean that the gate for *H+* is also closed. As far as this reviewer knows, nobody has actually demonstrated that H+ is occluded, even in the E2 state of SERCA.
Furthermore, the authors presume that protons enter the binding sites through a different pathway from that used for Ca2+ release, citing ref 26. However, if it does, can closure of the gate for Ca2+ really mean closure for the gate for H+? This seems a contradictorily statement as the authors designate that the E2·BeF3- state in Listeria Ca2+-ATPase as a proton occluded state (p.12). Apparent closure of the gate for Ca2+ on the extracellular side in a crystal structure seems insufficient for such a statement. One must keep in mind that a crystal structure merely provides a possible conformation in that particular state. It may not, however, represent the most populated conformation for that state. It is equally plausible that the E2·BeF3- complex takes a closed conformation for only a small fraction of the time. At this resolution it is simply not possible to determine if H+ occupies the binding site in the crystal structure. Furthermore, although it may be possible to show the gate is closed for Ca2+, it would be very difficult to show the gate is closed for H+. Thus, more experimental evidence is required to support that the structure represents a H+ *occluded* state.
The authors write in the Abstract "Structures with BeF3- mimicking a phosphoenzyme state reveal a closed state, which is intermediate of the outward-open E2P and the proton-occluded E2-P* conformations known for SERCA". In essence this statement is fine, although what "closed" means is still unclear to me. In Figure 1 (now Figure 2), the authors state that "LMCA1 structures adopt proton-occluded E2 states". This statement is a bit misleading, because, in E2·BeF3-, the lumenal (extracellular) gate can in fact be opened and closed, at least with SERCA. As the authors recognize (p.14), the BeF3- complex of SERCA can be crystallised in two conformations, one with the lumenal gate is closed (with thapsigargin) and the other with the gate open; yet, they write "In SERCA, the calcium-free BeF3 -complex adopts an outward-open E2P state,..." p.8). This is for lumenal (extracellular) Ca2+, not for H+. Further evidence is required to establish that the extracellular gate of LMCA1 is fixed in a closed position for H+ in E2·BeF3-. Again more experimental evidence is required to support that E2·BeF3- is a H+ occluded state.
The underlying challenge is that it is incredible difficult to demonstrate proton occlusion experimentally: The protons are invisible in most crystal structures and experimental variation of the H+ concentration affects many parts of the molecule. This means that it is not possible to get the same level of evidence for occlusion as for e.g. Ca2+, and as the reviewer states this has also not been achieved for other pumps.
This does not mean that it is impossible to deduce information about protonation states and H+ pathways from a crystal structure. A buried side chain is thus unlikely to be charged unless it is paired with a neutralizing charge, and we can thus reasonably deduce protonation states from structure-driven pKa prediction. Second, it is known from functional studies that LMCA1 and other Ca2+-ATPases counter-transport protons, so some of the transport site residues must be protonated. We think it is reasonable to interpret the crystal structure in terms of the most likely residues involved in proton counter-transport. <br />
We agree with reviewer #3 that the crystal structure only represent a single (likely highly populated) conformation. However, this criticism is equally true of any other crystal or cryoEM structure, and does not prevent such structures from being useful. It is tricky to precisely map proton access as they can be relayed via protonatable residues, i.e. “proton wires”. It is unlikely that any experimental method would unambiguously probe proton accessibility, and molecular dynamics would be unlikely to be conclusive due to the coupling between dynamics and protonation state. As absolute proton occlusion is difficult to demonstrate, we think it is more useful to think in terms of relative rates of proton exchange. All other things being equal, a residue that is fully exposed to the solvent will exchange protons more rapidly than a residue that relies on proton relaying or breathing motions in a protein. In this context, it is reasonable to consider this state a proton occluded-state.
To reflect this, we have edited the manuscript as follows:<br />
We have edited the “Results” section so it focuses on the immediate structural interpretation, i.e. pKa prediction and comparison of ion pathways. Discussion of the mechanisms that strays from the immediate structural interpretation has been moved to the “Discussion” section as proposed. The section headers have been updated to reflect this so now they discuss “Ion pathways and binding sites” and “Transport site protonation” rather than the “Mechanism of proton counter-transport”. Overall, we have softened the language describing proton occlusion to reflect that this is our best current interpretation and not established fact. Furthermore, we have qualified the statement about what a proton occluded state is:
“It should be noted that occlusion has a slightly different meaning for protons than e.g. Ca2+, as it is difficult to experimentally demonstrate proton occlusion. Furthermore, a crystal structure only provide a single snapshot of a protein and it is likely that protein dynamics will allow proton access to a certain extent. In the following, we describe a state as proton occluded, if it the ion binding site is closed to direct solvent access”
The authors write that "SERCA has two proposed proton pathways: a luminal entry pathway [26] and a C-terminal cytosolic release pathway [27] (p. 9). One has to be careful here, as the luminal entry pathway has not been experimentally confirmed in SERCA. The authors write that "The luminal proton pathway has been mapped to a narrow water channel ... [26]. But since the pathway is not confirmed in SERCA I don't think it can be used to justify that the corresponding part of LMCA1 is mainly hydrophobic and that protons cannot enter through this pathway.
As discussed above, experimental confirmation of a proton pathway is really tricky, but the structural comparison of the different residues in this region is unambiguous. We think it is reasonable to keep this comparison in the manuscript, but have rephrased the it to the “proposed” luminal proton pathway, and rephrased to remove the word “mapped”, which suggests experimental verification.
The description on the exit pathway for H+ also needs clarification. They describe (p. 10; first line) "In SERCA it consists of a hydrated cavity...[27]. ... M7 in LMCA1 further blocks the pathway ... and LMCA1 therefore does not appear to have a C-terminal cytosolic pathway either" and rationalize that "This may explain why no distinct proton pathways are required in LMCA1". I think it should be made clearer that this is a *proposal* rather than an established *fact*.
This section has been re-phrased and merged into the discussion.
As H+ release takes place in the E2 to E1 transition the authors state that the E2·BeF3- structure of LMCA1 is different from that of SERCA. However, I don't think they can confidently make such statements without E1 and E2 structures of LMCA1. Furthermore, these descriptions (discussion) should not be in the "Results" section. As they conclude that LMCA1 use the Ca2+ release pathway, which is assumed to be the same as that in SERCA (even though no Ca2+ release pathway is visualised in their crystal structures), for H+ entry, why does SERCA not use the same pathway? I think experimental evidence is required for a proposal that H+ binds to E309 from the cytoplasmic side.
Proton release likely takes place in the E1 state, not the transitions. Getting a crystal structure of this state would be great, but falls outside the scope of a revision. We compare our crystal structures of LMCA1 to the E2 crystal structures of SERCA, and they are clearly more similar to the E2-AlF state (see new Table S2). This is a straight forward alignment of a protein to its closest homologue with an available structure, so we think it is fair to keep this in the “Results”.
As this paper focus on LMCA1 and not SERCA, we think that both protonation of E309 and ion pathways in SERCA fall outside the scope of the manuscript except as a reference for LMCA1. However, as SERCA has additional pathways it will presumably be a question of kinetic competition.
The issue of proton counter-transport is dealt with above.
Additionally all the minor comments from reviewer #3 have been dealt with in the updated version 2 of the manuscript.