Structure of Escherichia coli respiratory complex I reconstituted into lipid nanodiscs reveals an uncoupled conformation

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1. 

   eLife

   Jul 27, 2021

   Author Response:

   Reviewer #2 (Public Review):

   [...] 1) A weakness of the paper is the disruption of the complex during cryoEM grid preparation resulting in about half of the observed particles missing the membrane arm and likely also contributing to the disorder and biased orientation seen in the intact complexes. This leads to poor density in the membrane arm for all of the intact complex I structures presented and large variations in the local resolution of the membrane arm focused refinement.

   Purified E. coli complex I has always been known to be labile in particularly at the junction of peripheral and membrane arms (https://pubmed.ncbi.nlm.nih.gov/12637579/).

   Air-water interface likely plays a role in disrupting the complex in addition to other possible causes. Indeed, the dissociated arms, preferred particle orientation, and low …

   Author Response:

   Reviewer #2 (Public Review):

   [...] 1) A weakness of the paper is the disruption of the complex during cryoEM grid preparation resulting in about half of the observed particles missing the membrane arm and likely also contributing to the disorder and biased orientation seen in the intact complexes. This leads to poor density in the membrane arm for all of the intact complex I structures presented and large variations in the local resolution of the membrane arm focused refinement.

   Purified E. coli complex I has always been known to be labile in particularly at the junction of peripheral and membrane arms (https://pubmed.ncbi.nlm.nih.gov/12637579/).

   Air-water interface likely plays a role in disrupting the complex in addition to other possible causes. Indeed, the dissociated arms, preferred particle orientation, and low protein concentration (~0.1 mg/ml) used to produce grids with high particle density all indicate that reconstituted complex I does interact with air-water interface. While disruption and denaturation of protein complexes on air-water interface has been well documented, (https://pubmed.ncbi.nlm.nih.gov/3043536/, https://pubmed.ncbi.nlm.nih.gov/30932812/ ), we are not aware of examples where air-water interfaces caused higher mobility of a complex or induced a stable conformation, different from the one in bulk solution. Therefore, we think that air-ware interface is neither the cause of the observed high arms mobility nor of their relative rotation.

   Preferential orientation was observed in the cryo-EM studies of most complex I homologs (Gutiérrez-Fernández et al., 2020; Parey et al., 2019; Zhu et al., 2016) as well as of other proteins, suggesting that adsorption of complex I on air water interface is a common phenomenon. In this case it is not clear why relative movement of the arms observed in all the structurally characterized complex I homologs is not due to the air-water interface, but in the case of E. coli complex it is.

   To provide additional support to our interpretation of the structural data we purified complex I in detergent LMNG, showed that it catalyzes redox reactions and solved its structure to resolution of 6.7 Å (Figure 6 and corresponding figure supplements). Because cryo-EM grids had to be prepared at a protein concentration of 2-3 mg/ml and the particles displayed nearly homogeneous distribution of orientations, we conclude that the interaction with the air-water interface was reduced. Still, the complex assumes a very similar, or even somewhat more uncoupled conformation and the relative mobility of the arms remained comparable to that in the nanodisc-reconstituted complex reconstructions. These data allow us to rule out the air-water interface and reconstitution of the protein into lipid nanodiscs as the possible causes of the high mobility and the unusual relative position of the arms.

   The corresponding modifications were added to the manuscript on lines 372-382:

   “To better understand the reasons for the observed uncoupled conformation and the missing density for HTMH1, we purified E. coli complex I in detergent LMNG, showed that it can catalyze redox reactions (Figure 6 - figure supplement 1) and solved its structure to resolution of 6.7 Å (Figure 6 - figure supplement 2). The detergent-solubilized complex also displays high relative mobility of the arms (Figure 6 - figure supplement 3) and has uncoupled conformation (Figure 6). Its peripheral arm is rotated even further away from the expected coupled state position than in the nanodisc-reconstituted structures. Both the cryo-EM sample preparation conditions and more homogeneous distribution of particle orientations indicate that interaction of the complex with air-water interface was significantly reduced when compared with the complex in nanodiscs. This allows us to conclude that neither air-water interface nor reconstitution into nanodiscs cause the uncoupled conformations.”

   It is not very clear what referee means by “poor”, when referring to the focused density of the membrane arm. The density corresponds well to the reported resolution of 3.7 Å. Indeed, it is in a stark contrast with the quality of the density obtained for the peripheral arm at 2.1 Å resolution. Given high mobility of the membrane arm it had to be refined essentially independently of the peripheral arm which remains still challenging for a ~200 kDa membrane protein without water-soluble domains in lipid nanodiscs. The density is heterogeneous as clearly stated at the beginning of the section “Structure of membrane arm” from line 264:

   “The model of complete membrane arm, including the previously missing subunit NuoH (Efremov and Sazanov, 2011), was built into the density map with local resolution better than 3.5 Å at the arm center and approximately 4.0 Å at its periphery (Figure 1A, Figure 1 - figure supplement 4).”

   Finally, for most complex I homologs the resolution was gradually improved over several years, as reflected in multiple publications of essentially the same structures. In contrast, no high-resolution structure information was available for the intact E. coli complex I until now. Therefore, it would be unreasonable to expect the complete structure to be solved at resolution of 2 Å at once.

   The resolution of the membrane domain in reconstructions of complete complex I is indeed lower due to high flexibility of the complex and the fact that refinement naturally focuses on more stable peripheral arm that does not have heterogeneous nanodisc around and that contains Fe-S clusters enhancing particle alignment power. Still, these conformations clearly resolve the interface between subunits albeit at lower resolution.

   This fact was also clearly stated at the beginning of results section lines 102-106:

   “Three conformations of the entire complex were reconstructed to average resolutions between 3.3 and 3.7 Å (Figure 1 - figure supplement 4) resolving the interface between the arms; however, due to high-residual mobility of the arms, the antiporter-like subunits were resolved at below 8 Å (Figure 1 - figure supplement 4).”

   1. A weakness of the paper is the disorder of important functional regions of the complex, namely the NuoH TMH1, whose disorder is unique to these nanodisc E. coli structures, and the NuoA TMH1-TMH2 loop. As the NuoH TMH1 forms part of the entry to the quinone tunnel of the complex, its absence in the structure leads to concerns regarding the function of the nanodisc preparation. Its absence it curious as this suggests flexibility of the helix, as pointed out by the authors, but the authors also state that there is not enough room in the nanodisc to accommodate this helix (given the visible density for the lipid and membrane scaffold protein). These observations suggest denaturation or unfolding in this region of the complex as opposed to simple flexibility.

   According to the usual definition of complex I activity our preparation in nanodiscs is active. We complemented our data with additional measurements and included NADH:DQ assays (see next point) that also indicate that our preparation is active. Additional 3D reconstruction of E. coli complex I that we obtained for protein solubilized in LMNG does resolve HTMH1 and its environment appears to be more similar to other detergent-solubilized structures of complex I homologues. At the same time, the helices around HTMH1 appear to be more tightly packed and more curved than in the nanodiscs which may reflect suppressed dynamics and distorted protein conformation. Most importantly, the overall conformation of the complex remains nearly the same and still corresponds to what we call the uncoupled conformation. That of course does not allow us to say where HTMH1 is positioned within the nanodisc, but it does enable us to conclude that the local changes in the vicinity of HTMH1 do not influence the global conformation of the complex.

   The additional structure is not described on lines 383-388:

   “The HTMH1 helix is resolved in the detergent-solubilized complex (Figure 6A). Its density is weaker than that of the surrounding helices and it is strongly bent (Figure 6B). Simultaneously, HAH1 takes the conformation resembling other complex I homologs while ATMH1 bends towards the arm core. The arrangement of helices in detergent-solubilized reconstruction appears to be more compact and more bent than in the lipid environment which may restrain the otherwise more flexible HTMH1.”

   In the revised discussion the environment of HTMH1 is described more clearly on lines 426-433:

   “The absence of HTMH1 density in nanodiscs, but not in detergent, is another unique feature of E. coli complex I. HTMH1 is exposed to the lipid environment and the width of the nanodisc next to HTMH1 is similar to other regions around the membrane arm (Movie 1). Moreover, homology modelled HHTM1 fits the empty space without steric clashes suggesting that HHTM1 is dynamic rather than displaced or unfolded. By comparing the detergent-solubilized and reconstituted complexes we can conclude that position and dynamics of this helix is neither the cause of the uncoupled conformation nor of the high relative mobility of the arms.”

   Disorder of ATMH1-TMH2 loop is not unique to E. coli complex I but also observed in some conformations of ovine complex I PDB 6zkd, 6zke, 6zkf.

   1. Unfortunately, the NADH:Q1 functional data do not fully address these concerns at Q1 is far more soluble that the native Q8 substrate of the complex. Although the Q1 activity is sensitive to the inhibitor Piericidin A, which clearly demonstrates that the Q1 reduction is occurring in the native quinone binding site as Piericidin A binds specifically at that site, this does not preclude the possibility of Q1 accessing this binding site via a different path. In fact, the structures indicate that given the flexibility in the connection between peripheral and membrane arms of the complex, the quinone binding site is likely open to the cytoplasm. This leads the authors themselves to conclude that the structures presented are likely disrupted/uncoupled states in which the energy converting mechanism of the complex is not likely possible.

   To address the raised concern, we have measured the activity of complex I in nanodiscs with less soluble decylubiquinone (DQ) as well as its inhibition. Small amounts of LMNG was used to increase the DQ solubility. Our results have confirmed that E. coli complex I in nanodiscs is active and the NADH:DQ activity is sensitive to piericidin A (see the modified Figure 1-figure supplement 2). We have also remeasured the Q1 activity and its inhibition which showed lower values than previously, due to a flaw in the activity measurements reported in the original submission (qualitatively, the results remained unchanged). Moreover, we have observed a similar activity results with somewhat higher values for E. coli complex I in LMNG (Figure 6-figure supplement 1). These data demonstrate that in the reconstituted complex I quinone analogues can enter the Q-site through the membrane. It is worth noting that due to extremely low solubility of longer quinones, including native ones, they are not used for activity measurements in purified preparations.

   Regarding the complex I conformation, we do think our reconstruction represents uncoupled state which is not able to pump protons (as states in the title). We have improved the clarity of this point throughout the manuscript including the discussion lines starting from the line 412.

   “The high mobility of the interfacial regions and the relative rotation of the arms disrupts conserved interfacial interactions and exposes Q-cavity to the solvent (Figure 5A). This differentiates E. coli complex I from its structurally characterized homologs in which the Q-cavity is sealed from the solvent. Thus, we interpret the observed conformation as an uncoupled state.”

   And from line 469: “We also observed the relative rotation of the membrane and peripheral arms disrupting the conserved interface and trapping the complex in an uncoupled conformation. Whether this conformation is biologically relevant or is a result of protein purification is to be clarified by further research.”

   1. A weakness of the paper is the building of atomic models into regions of the map which do not contain sufficient detail to warrant atomic models. This is particularly the case for the intact models of complex I as well as the membrane arm focused maps and results in low map-model correlations (0.58-0.71). The models were clearly highly restrained during refinement, resulting in good geometry, as is necessary for low resolution regions. But being able to restrain the geometry is not sufficient for placing atoms into regions where the density is weak or absent. If additional information was used in building/constraining the model, such as the X-ray structure, the regions of the model that are biased towards the X-ray structure model needs to be made clearer. Also, in several places in the membrane arm map residues bulge out of the density (side chain and main chain) leading to possible frame shifts with respect to the match between subsequent residues in the model and the map (see NuoM Ile168 for example).

   A large part of the membrane domain has been solved using X-ray crystallography to resolution of 3.0 Å which was used as a starting model for model building, therefore we don’t think there are register shifts in our model. We used standard setting for model refinement in phenix_refine. Our building and refinement procedure has been described in fine details in the original submission, see from line 674:

   “For the membrane domain, the previously obtained E. coli model (PDB ID: 3RKO) was real-space-refined in PHENIX. The missing NuoH subunit was homology-modelled using the T. thermophilus structure (PDB ID: 4HEA) in Coot 0.9. The final model was obtained after several rounds of manual rebuilding and real-space refinement using standard parameters with Ramachandran restrains, secondary-structure restrains applied to the NuoL TMH9-13, without ADP restrains, and with the optimized nonbonded_weight parameter. To generate the model of the complete complex I, the separate peripheral and membrane arm structures were combined and the missing parts at the interface (Table 2) were built manually. As the density of NuoL and NuoM was very poor in all the resolved full conformations, these subunits were subjected to rigid-body refinement in PHENIX, whereas the others were subjected to real-space refinement with minimization_global, local_grid_search, morphing, and ADP refinement. Ramachandran, ADP, and secondary-structure restrains were used. After manual rebuilding in Coot, real-space refinement of the full complex was performed with standard parameters and restrains.”

   To improve clarity, we added a following sentence to the Results section from line 116:

   “Using the resulting maps, atomic models of the peripheral and membrane arms have been built. The entire E. coli complex I was modelled by fitting models of the arms and extending additionally resolved loops and termini. Due to limited resolution, the antiporter-like subunits were refined as rigid bodies.”

   The model has been improved and side chains with absent density were truncated to C position.

   The density for focused refinement density of the membrane fragment is relatively week, but of sufficient quality to allow building side chains for most of the map. It even visualizes lipid densities (not described in the manuscript). Such weaker densities are common for small membrane proteins. While fully usable for model building, they naturally result in lower model map FSC and consequently, in lower real-space correlation. In addition, real-space correlation is lower when the map is heterogeneous, and it strongly depends on the way the heterogeneous map has been filtered. Therefore, lower cross correlations do not necessarily mean that the model fit is poor. In our case they reflect weaker signal to noise of the density. Model-map FSCs (Figure 1 figure supplement 4) are more informative than a single number and show that model-map cross correlations remain above 0.5 for the complete resolution range for all models.

   1. A weakness of the paper is that several specific claims are made about the positions of side chains but, when investigated, the density for those side chains is poorly resolved. An example of this is NuoH Lys274, which is in a low-resolution region of the map and although is fit as well as possible must be considered low confidence given the local resolution (nearby residues Phe277 and Phe282 have almost no side chain density for example).

   At lower resolution, a presence of residues density strongly depends on their mobility. Well-ordered residues may have well-defined densities while others, even in the proximity, may have a poor density. In the case of Lys274, there is a clear density for the side chain, its position makes chemical sense, and it is hydrogen-bonded to the backbone oxygen of Gly258. In fact, if examined closely, this is also the only meaningful position for Lys274 side chain. At the same time, the conformations of Phe277 and Phe282 are not restrained by interactions with other residues in their vicinity which is likely why their densities are weaker.

   1. A weakness of the paper is that the conformational changes seen between the membrane and peripheral arm of the complex in the different 3D classes are difficult to interpret. It is unclear if they are mechanistically significant or, perhaps more likely given the amount of broken complex observed, due to partial disruption of the complex before it completely breaks apart.

   As we discussed above, the observed multiple conformations are not due to the complex disruption. It is not very clear what the reviewer means by ‘difficult to interpret’. Many conformations of the peripheral and membrane arms observed for the complex I homologues are likely not mechanistically meaningful per see, but rather reflect overall flexibility of such a large complex. Here, our goal was to describe our structural data as accurately as possible which resulted in several resolved conformations.

   We do think they all represent the uncoupled complex I, in this respect they do not have different mechanistic meanings. However, they do permit us to understand how the arms move relative to each other and what degree of freedom exists between them.

   1. A strength of the paper is the interesting and original mechanistic proposal put forward by the authors. But a weakness is that it is unclear how this proposal stems from the structural data presented. Also, the arguments presented are difficult to follow in their current form and warrant a more detailed discussion with the requisite thermodynamic treatment. This may warrant a more complete discussion in an appendix or unless the authors can more convincingly show how the data presented in the paper suggests their proposed mechanism perhaps a separate review article. Furthermore, the proposed mechanism, as presented would make a simple prediction that in the absence of NuoM and NuoL (or equivalent subunits in other species) complex I would not pump any net protons. Experiments that are relevant to this prediction have been done in E. coli (NuoL deletion) and Y. lipolytica (nb8m deletion that results in loss of both NuoM and NuoL subunits). See https://pubmed.ncbi.nlm.nih.gov/21417432/ and https://pubmed.ncbi.nlm.nih.gov/21886480/. In both cases the complex is still able to pump protons. The behavior of the NuoL deletion in E. coli is reconcilable with their proposed mechanism as NuoM is still present, however, the case of the nb8m deletion in Y. lipolytica is more difficult to reconcile with their proposed mechanism. The authors would need to address these experiments in order to include their proposed mechanism.

   The description of the mechanism has been modified. It is very briefly outlined in the main text along with the Figure 7 and more detailed description, including thermodynamic considerations, is moved to the supplementary text. We have also explained more clearly how the model stems from the experimental data on line 435:

   “The absence of a continuous proton-translocation pathway between the Q-site and subunit NuoN, as well as high flexibility of the peripheral arm interface are not consistent with the recently proposed coupling mechanisms relying on specific movements of the interfacial loops (Cabrera-Orefice et al., 2018; Kampjut and Sazanov, 2020). This led us to ask whether a coupling mechanism consistent with known complex I properties, but without the movements of interfacial loops is conceivable.”

   Furthermore, we state that at this point this is a hypothetical mechanism.

   Supplementary data describing mechanism in more details now also includes the discussion of both papers mentioned by the reviewer from line 1368.

   “Experiments with engineering E. coli complex I lacking subunit NuoL and Y. lipolytica complex I lacking homologs of subunits NuoM and NuoL (Dröse et al., 2011; Steimle et al., 2011)(Dröse et al., 2011; Steimle et al., 2011) (correspond to n=2 and 1, respectively) both suggested that the engineered complexes were active and for both constructs stoichiometry was estimated as 2H+/2e-. While NuoL deletion experiments support our model, the NuoL/M deletion clearly contradicts it. Both experiments should be interpreted cautiously, however. Results of NuoL deletion for E. coli complex I were not reproducible (Verkhovskaya and Bloch, 2012). In the case of Y. lipolytica, the homologs of NuoL/M dissociated from the complex along with another 11 subunits upon deletion of supernumerary subunit NB8M located at the tip of NuoL (Zickermann et al., 2015). Since the proton-translocating modules were not deleted per se, the presence of contaminating amounts of assembled complex I in the preparations that generated observed proton pumping cannot be completely excluded. It is important to note that mutation of the conserved ionizable residues on the interface between NuoN and NuoM, i.e. ME144 (Torres-Bacete et al., 2007) or its counter ion NK395 (Amarneh and Vik, 2003), result in a completely inactive complex I suggesting that dissociation of subunits NuoL/M also should render complex I inactive (Verkhovskaya and Bloch, 2012).”

   The main problem with these experiments that that they have never been reproduced by other laboratories and are not completely consistent with the mutagenesis data. Deletion of subunits may also result in distinct pumping behavior of the remaining subcomplex. For example, it was shown for the bovine complex I that it can translocate Na+ ions in the deactive state (https://pubmed.ncbi.nlm.nih.gov/22854968/).

   Appraisal of whether the authors achieved their aims, and whether the results support their conclusions:

   1. Overall, despite the many strengths of this paper detailed above it is unclear whether the authors achieved their goal of a structure of functional E. coli respiratory complex I reconstituted in lipid nano-discs. It appears that under the current grid preparation conditions that the complex is under excessive stress resulting in partial denaturation and partial-to-complete dissociation. Given the clear biophysical data presented on the intactness of the complex in solution, this disruption likely occurs during grid preparation and further optimization of grid conditions may resolve this issue. With the current maps more work needs to be done to improve the map-to-model correlation and to clearly indicate the regions in the models where this correlation is low.

   Additional reconstruction of complex I solubilized in LMNG help us to exclude the interaction of the complex with water-air interface and its reconstitution into lipid nanodiscs as the causes of the relative subunit rotation and high flexibility between the arms. At this moment, whether the structure represents an artifact of purification or is a biologically-relevant state remains an open question. However, answering it goes beyond the current study and will require additional research. This is now explained in the discussion section.

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2. 

   eLife

   Jul 27, 2021

   Evaluation Summary:

   The manuscript reports the cryoEM structure of a functional E. coli respiratory complex I (proton-pumping NADH-ubiquinone oxidoreductase) reconstituted in lipid nano-discs. The reconstructions and models presented by the authors indicate interesting E. coli specific features of the complex, although there are some concerns about model accuracy. Overall this can be a major advance for the structure of this important respiratory complex from a key model organism.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

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3. 

   eLife

   Jul 27, 2021

   Reviewer #1 (Public Review):

   Energy in the form of ATP is key for any function of the cell. Most organisms have a series of protein complexes in their membrane that transport proton against a gradient using the redox reactions and this is then used by the enzymes called ATP synthases that generate ATP, the energy needed to sustain life. The first enzyme in this electron transport chain is called complex I and uses the oxidation of NADH to the reduction of ubiquinone, which is coupled to proton translocation. The unique shape of this complex had been realized from early electron microscopy studies and they can be divided into a soluble domain that comprises all the co-factors needed for electron transfer and the membrane domain that has modules for proton translocation. Depending on the organism, this complex can be compact with only the …

   Reviewer #1 (Public Review):

   Energy in the form of ATP is key for any function of the cell. Most organisms have a series of protein complexes in their membrane that transport proton against a gradient using the redox reactions and this is then used by the enzymes called ATP synthases that generate ATP, the energy needed to sustain life. The first enzyme in this electron transport chain is called complex I and uses the oxidation of NADH to the reduction of ubiquinone, which is coupled to proton translocation. The unique shape of this complex had been realized from early electron microscopy studies and they can be divided into a soluble domain that comprises all the co-factors needed for electron transfer and the membrane domain that has modules for proton translocation. Depending on the organism, this complex can be compact with only the core 14 subunits as found in many bacteria or decorated with a number of other subunits as found in eukaryotes. These additional subunits are implicated in the stability and regulation of the complex. With the advances in cryoEM, in the last few years there has been a flurry of structures from different organisms, the composition and arrangement of subunits, potential functional states and description of the mechanism. An important factor to consider is that these complexes are labile and the detergents which are used for purification can have a profound effect on the structure as well as any conclusion drawn from it. In the field of Bioenergetics, one of the key questions that have fascinated scientists for a long time is how the electron transfer is coupled to proton translocation and few different hypotheses has been put forward.

   Adding to the growing number of complex I structures, the manuscript by Kolata and Efremov discusses the structure of a complex I from Escherichia coli, a well-studied enzyme and amenable to easy genetic engineering, essential to verify proposed mechanisms. The key observations of the current work and future prospective are discussed below.

   The E. coli complex I is known to be labile and previous attempts to crystallize the whole complex had yielded a low-resolution structure of the membrane domain. The authors have overcome this by introducing a tag in the bacterial genome using CRISPR technology minimizing the number of steps needed for enrichment. The manuscript also addresses one of the concerns of the complex I structures i.e., effect of detergent environment by determining the structure of a mesophilic bacterial complex I in nanodisc. Despite the presence of the lipidic environment, substantial number of particles have only the soluble peripheral arm and only half the number of particles exist as full complex indicating the extreme instability of this complex. By extensive computational classification to address heterogeneity, the maps have been improved both for the full complex and the peripheral arm at a very high resolution (2.1 Å), allowing to build almost a complete protein model as well as a number of water molecules, key in understanding the mechanism. Differences in E. coli complex with structures of other enzymes are discussed, in particular the junction of the peripheral arm and the membrane domain and TMH8 in the NuoM subunit of the membrane domain. With all the structural and biochemical details, the authors propose a coupling mechanism that is different from those proposed previously and involves the idea that protons enter the ubiquinone cavity via the periplasmic or intra-membrane side (in organelles) and this is coupled to 3 protons being transported by the membrane domain in the opposite direction. Thus, for each reduction of ubiquinone 4 protons are translocated in two pumping cycles.

   While the mechanism is simple and elegant, the absence of a structure with bound ubiquinone (the cavity for ubiquinone is inferred from other structures) and with NADH, different states observed by classification, dissociation of the peripheral arm - all of which asks for some caution and is a caveat. Nevertheless, these structures are important and will be the platform for further studies (addition of NADH, ubiquinone, pH etc.,) and the proposal can be verified by further biochemical and computational studies.

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4. 

   eLife

   Jul 27, 2021

   Reviewer #2 (Public Review):

   Summary of what the authors were trying to achieve:

   Kolata and Efremov set out to achieve a cryoEM structure of functional E. coli respiratory complex I (proton-pumping NADH-ubiquinone oxidoreductase) reconstituted in lipid nano-discs by single particle cryo-electron microscopy (cryoEM).

   Strengths and weaknesses of the methods and results:

   A strength of the paper is that E. coli respiratory complex I is one of the most studied homologues of the enzyme with many important functional and mutagenesis studies published. For this reason, the complex I field has been anticipating the structure for some time.

   A strength of the paper is the production of a E. coli cell line harboring a Twin-Strep tag on the complex I subunit NuoF using the Crispr-Cas9 system. This allows the authors to develop a single step …

   Reviewer #2 (Public Review):

   Summary of what the authors were trying to achieve:

   Kolata and Efremov set out to achieve a cryoEM structure of functional E. coli respiratory complex I (proton-pumping NADH-ubiquinone oxidoreductase) reconstituted in lipid nano-discs by single particle cryo-electron microscopy (cryoEM).

   Strengths and weaknesses of the methods and results:

   A strength of the paper is that E. coli respiratory complex I is one of the most studied homologues of the enzyme with many important functional and mutagenesis studies published. For this reason, the complex I field has been anticipating the structure for some time.

   A strength of the paper is the production of a E. coli cell line harboring a Twin-Strep tag on the complex I subunit NuoF using the Crispr-Cas9 system. This allows the authors to develop a single step purification of the complex and will be very useful for any future work on E. coli complex I.

   A strength of the paper is the multiple methods used to test the integrity of the nanodisc reconstituted complex (i.e., size exclusion chromatography and mass photometry) and the functional assays demonstrating inhibitor sensitive NADH:Q1 oxidoreductase activity.

   A strength of the paper is the high-resolution structure of the peripheral (cytoplasmic) arm of the complex. This structure reveals several features unique to complex I and suggests different strategies for stabilization of the peripheral arm subunits have evolved in different lineages.

   A weakness of the paper is the disruption of the complex during cryoEM grid preparation resulting in about half of the observed particles missing the membrane arm and likely also contributing to the disorder and biased orientation seen in the intact complexes. This leads to poor density in the membrane arm for all of the intact complex I structures presented and large variations in the local resolution of the membrane arm focused refinement.

   A weakness of the paper is the disorder of important functional regions of the complex, namely the NuoH TMH1, whose disorder is unique to these nanodisc E. coli structures, and the NuoA TMH1-TMH2 loop. As the NuoH TMH1 forms part of the entry to the quinone tunnel of the complex, its absence in the structure leads to concerns regarding the function of the nanodisc preparation. Its absence it curious as this suggests flexibility of the helix, as pointed out by the authors, but the authors also state that there is not enough room in the nanodisc to accommodate this helix (given the visible density for the lipid and membrane scaffold protein). These observations suggest denaturation or unfolding in this region of the complex as opposed to simple flexibility. Unfortunately, the NADH:Q1 functional data do not fully address these concerns at Q1 is far more soluble that the native Q8 substrate of the complex. Although the Q1 activity is sensitive to the inhibitor Piericidin A, which clearly demonstrates that the Q1 reduction is occurring in the native quinone binding site as Piericidin A binds specifically at that site, this does not preclude the possibility of Q1 accessing this binding site via a different path. In fact, the structures indicate that given the flexibility in the connection between peripheral and membrane arms of the complex, the quinone binding site is likely open to the cytoplasm. This leads the authors themselves to conclude that the structures presented are likely disrupted/uncoupled states in which the energy converting mechanism of the complex is not likely possible.

   A weakness of the paper is the building of atomic models into regions of the map which do not contain sufficient detail to warrant atomic models. This is particularly the case for the intact models of complex I as well as the membrane arm focused maps and results in low map-model correlations (0.58-0.71). The models were clearly highly restrained during refinement, resulting in good geometry, as is necessary for low resolution regions. But being able to restrain the geometry is not sufficient for placing atoms into regions where the density is weak or absent. If additional information was used in building/constraining the model, such as the X-ray structure, the regions of the model that are biased towards the X-ray structure model needs to be made clearer. Also, in several places in the membrane arm map residues bulge out of the density (side chain and main chain) leading to possible frame shifts with respect to the match between subsequent residues in the model and the map (see NuoM Ile168 for example).

   A weakness of the paper is that several specific claims are made about the positions of side chains but, when investigated, the density for those side chains is poorly resolved. An example of this is NuoH Lys274, which is in a low-resolution region of the map and although is fit as well as possible must be considered low confidence given the local resolution (nearby residues Phe277 and Phe282 have almost no side chain density for example).

   A weakness of the paper is that the conformational changes seen between the membrane and peripheral arm of the complex in the different 3D classes are difficult to interpret. It is unclear if they are mechanistically significant or, perhaps more likely given the amount of broken complex observed, due to partial disruption of the complex before it completely breaks apart.

   A strength of the paper is the interesting and original mechanistic proposal put forward by the authors. But a weakness is that it is unclear how this proposal stems from the structural data presented. Also, the arguments presented are difficult to follow in their current form and warrant a more detailed discussion with the requisite thermodynamic treatment. This may warrant a more complete discussion in an appendix or unless the authors can more convincingly show how the data presented in the paper suggests their proposed mechanism perhaps a separate review article. Furthermore, the proposed mechanism, as presented would make a simple prediction that in the absence of NuoM and NuoL (or equivalent subunits in other species) complex I would not pump any net protons. Experiments that are relevant to this prediction have been done in E. coli (NuoL deletion) and Y. lipolytica (nb8m deletion that results in loss of both NuoM and NuoL subunits). See https://pubmed.ncbi.nlm.nih.gov/21417432/ and https://pubmed.ncbi.nlm.nih.gov/21886480/. In both cases the complex is still able to pump protons. The behavior of the NuoL deletion in E. coli is reconcilable with their proposed mechanism as NuoM is still present, however, the case of the nb8m deletion in Y. lipolytica is more difficult to reconcile with their proposed mechanism. The authors would need to address these experiments in order to include their proposed mechanism.

   Appraisal of whether the authors achieved their aims, and whether the results support their conclusions:

   Overall, despite the many strengths of this paper detailed above it is unclear whether the authors achieved their goal of a structure of functional E. coli respiratory complex I reconstituted in lipid nano-discs. It appears that under the current grid preparation conditions that the complex is under excessive stress resulting in partial denaturation and partial-to-complete dissociation. Given the clear biophysical data presented on the intactness of the complex in solution, this disruption likely occurs during grid preparation and further optimization of grid conditions may resolve this issue. With the current maps more work needs to be done to improve the map-to-model correlation and to clearly indicate the regions in the models where this correlation is low.

   Likely impact of the work on the field, and the utility of the methods and data to the community:

   As the first structure of respiratory complex I from the important model organism E. coli this work will have a big impact on the field. This is due to the history of studies on complex I that have been performed using this organism. The structure of the peripheral arm presented here is itself a major advance for this field and presents several important new insights into the evolution of complex I in different lineages. Due to the problems outlined above the structure of the membrane arm and proposed mechanism are more difficult to evaluate and if the limitation of the models are not made more clear this could lead to misinterpretation by non-experts in the structural biology.

   Read the original source
5. 

   Version published to 10.7554/elife.68710 on eLife

   Jul 26, 2021
6. 

   Version published to 10.1101/2021.04.09.439197v1 on bioRxiv

   Apr 10, 2021