Peer review of: ABCG2 transports anticancer drugs via a closed-to-open switch (2020)

Reviewers' comments: 
 
Reviewer #1 (Remarks to the Author): 
 
The paper by Orlando and Liao presents some exciting new cryo-EM data on the drug binding site 
within the central cavity of the multidrug pump ABCG2. 
 
Two different structures are presented; the first obtained in the absence of drug reveals a highly 
unexpected conformation particularly with respect to the transmembrane domains, in which TM 
helices 5 and 2. This novel TMD arrangement for ABCG2 was supported by biochemical 
experiments using conformation-sensitive antibody binding and disulphide cross-linking. The 
second class of structure are drug-bound structures: notably the combination of the approaches 
enables the authors to discriminate between true substrate substrates (mitoxantrone and SN38) 
and transporter inhibitors that bind at the same site (imatinib). The authors present a compelling 
model to encapsulate this, and I think this paper would be of great interest and relevance to the 
ABC transporter community. 
 
Major: 
The results do not give any indications of the number of independent experiments and technical 
repeats. This is a really surprising omission which is of the highest priority to rectify. 
 
Lines 440-445. Can the authors provide some clarity regarding the final step of their purification? 
The protein is incubated with Mg.ATP prior to final HPLC analysis. For an “apo” structure this 
seems to be a very strange final step as it could introduce conformational heterogeneity into the 
preparation. 
 
Lines 320-322 and Lines 448-450. The authors use concentrations of drugs in their EM that are so 
much higher than one would expect to see (75 microM). I would expect affinities of these drugs to 
be in the sub microM range. Some comment here is needed. This becomes relevant when 
considering their suggestion that drugs provide a bias in the conformation of ABCG2; have the 
authors tested if the relative proportions of the two predominant conformations (lines 254) are 
sensitive to the applied drug concentration? One would fully expect this to be the case. Or that the 
relative proportions of the two predominant conformations (lines 254) are sensitive to the duration 
of drug incubation? 
 
Lines 154-171, 536-552; Figure 2. The cross-linking data described in this section is evidence for 
the sampling of the unusual conformation in figure 1. I do wonder why the work was performed in 
cells rather than membranes – one might expect that in membranes the cross-linking in the 
presence of drugs was much more substantial as there would be no drug export, which I suspect is 
why the percentage cross-linking is low. Were time courses performed for the cross-linking 
experiments? This might be relevant as kinetic information might be useful for deriving relative 
apparent drug affinities. 
 
Lines 185-215. I really liked the flow cytometry data provided which is genuinely elegant, but the 
ATPase assay data is less so. The expectation for an inhibitor is that ATPase activity would be 
decreased in a dose-dependent manner, and this is observed for Ko143 and for imatinib. For the 
two substrates (SN38 and mitoxantrone) I think the ATPase assay data are ambiguous. I would 
expect dose-dependent stimulation not inhibition. The authors assert that “this was consistent with 
that obtained for E1S” (estrone-1-sulphate; reference quoted 24) but comparison of the current 
paper Figure S1D with the figure S2 in the cited reference 24 (Manolaridis et al., 2018) does not 
support this. E1S shows a clear dose-dependent stimulation of ATPase activity from which an EC50 
concentration could be derived. The current ATPase data does not fully support the authors 
narrative here, although it does provide evidence that imatinib is an inhibitor. 
 
Lines 204-220 with no values for “n” and no evidence of experimental variability the data in Figure 

S1E should be removed and the test should be modified as well as there is no evidence of 
thermostabilisation as presented. 
 
Minor: 
From a narrative point of view I think the authors need to be clear about previous structures and 
their own structure throughout the paper. For example on line 107 the phrase “in the closed 
conformation” should read “in our apo-closed conformation” so that the reader knows exactly 
which structure is referred to. This is true elsewhere in the paper with respect to both their own 
structure and previous Locher group structures (e.g. lines 151-152). Please can the authors use 
the same terms for any references to these previous structures. I would prefer to see the pdb 
codes given in parenthesis to help the reader here. Figure legends would benefit from the same 
degree of clarity. 
Line 111: I cannot see the residue Met541 in Figure 1D – can the authors check this. 
Line 147-15: can the authors provide a supplementary table listing the predicted distances 
between the two residue pairs across their apo-closed structure, the Locher inward open structure 
and the Locher ATP-bound structure. This would really help the reader see that the predictions 
about residues being “too distant” are valid 
 
Lines 340-350. In the discussion the authors attempt explanation of their data with respect to the 
well described R482 residue but there are also publications describing mutation to M549, F545 and 
M523 that may be relevant here. 
 
 
 
 
 
Reviewer #2 (Remarks to the Author): 
 
Orlando et al., report the cryo-EM structures of the human ABC transporter ABCG2 in apo and 
three different anti-cancer drugs bound states (imatinib, mitoxantrone, and SN38). While the 
structures of human ABCG2 in fab, inhibitors and ATP bound states had been described previously 
(Taylor et. al 2017, Jackson et. al 2018, Manolaridis et. al 2018), the new ABCG2 apo structure 
described here reveals a novel closed conformation. Compared to previous ABCG2 structures in 
the inward-open state, both of TM2 and TM5 of ABCG2 in closed state exhibit significantly distinct 
conformations. Using site-directed disulfide crosslinking, Orlando et al. experimentally show that 
this closed conformation of ABCG2 exists in vivo and thus represents a functional relevant state. 
The structures of ABCG2 bound with three different anti-cancer drugs determined in this work 
adopt similar inward-open conformations as those previously observed in the structures of ABCG2 
with inhibitors bound. Based on the biochemical and structural studies, the authors propose that 
imatinib is more effective than another two anti-cancer drugs in stabilizing the inward-open 
conformation of ABCG2. 
 
Overall, the cryo-EM and biochemical works looks solid, and are of high technical quality. By 
elucidating the structure of ABCG2 in a novel closed conformation, this manuscript substantially 
advances our knowledge and understanding of the transporting mechanism of ABCG2, and provide 
structural implications for other members in ABC transporter family. The structures of ABCG2 with 
three different anti-cancer drugs bound help understanding how these drugs are transported out 
from the cancerous cells, which will facilitate the design of better drugs for chemotherapy. 
Considering these, this work can potentially make big impact on the field of ABC transporter and 
chemotherapy. 
I have only minor comments: 
(1) The binding modes between ABCG2 and either of three anti-cancer drugs are similar. Only one 
drug molecular is bound approximately at the 2-fold symmetrical axis of ABC2G dimer. Therefore, 
the overall structure ABCG2 with one drug bound is actually related by a pseudo C2 symmetry. 
The particle misalignment caused by the pseudo symmetry will definitely affect the structural 

determination of the drug bound ABCG2 structure. Under this circumstance, the accuracy of 
modelling a small compound might be poor. My sense is that the overall fitting of these 
compounds is relatively accurate, but the authors should briefly discuss the degree of uncertainty 
in modeling the drug, owing to the pseudo symmetry, in the main text. 
 
(2) The author claim that the imatinib is more effective than another two drugs in the stabilization 
of the inward-open conformation of ABCG2. However, the cryo-EM map of ABCG2 with imatinib 
bound was solved at relatively lower resolution, as compared with other cryo-EM maps. Could 
authors provide an explanation? I notice that the data collection strategy for ABCG2/imatinib 
sample is different from those used in other samples. Could this be the reason? 
 
(3) The workflows of particle classification for 4 different dataset are quite different. The authors 
should elaborate the procedures of imaging processing for each dataset in the method section. 
 
(4) The author should describe how the 3D initial model was generated. 
 
(5) The authors should show the representative 2D class averages at least for one of the 4 
datasets. 
 
(6) The author should describe how the FSC between model and map was calculated. 
 
(7) The title of Supplementary Figure 7: “Cryo-EM Data Analysis of ABCG2-mitoxantrone” should 
be changed to “Cryo-EM Data Analysis of ABCG2-MXN” to be consistent with the title of 
Supplementary Figure 6.