The C-terminal region of the motor protein MCAK controls its structure and activity through a conformational switch

Thank you for sending your work entitled “The C-terminal region of MCAK controls its structure and activity through a conformational switch” for consideration at eLife. Your article has been evaluated by John Kuriyan (Senior editor) and two reviewers. The editor has concluded that the paper may become suitable for publication in eLife if the issues raised by the referees can be addressed suitably. The reviewers, who also reviewed your original submission, have discussed their concerns with each other and the editor, and the review report merges all of the comments. The review report is appended below.

Review:

The authors present the crystal structure of the motor domain of the microtubule depolymerase MCAK in complex with a C-terminal peptide. The structure reveals that this peptide promotes dimerization of the motor-domain and regulates the conformation of MCAK's neck linker region. The structural data are well confirmed by biophysical methods and mutant studies.

The second part of the paper uses this structure to work out the role of the C-terminal peptide. The authors use covalent attachment of the C-terminal peptide to the motor domain to show that C-terminal peptide blocks MT binding. They then use a mutant (S715E) in which the C-terminal peptide can't bind. This leads to a high affinity interaction with the microtubule and decoration along the MTs length. The authors use their data to support a model in which the C-terminal peptide reduces the affinity of the MCAK motor domain for the lattice of the microtubule, allowing it to diffuse to the ends where its activity is required.

Overall the data are better presented in this draft and the story is clearer. The authors have added some nice experiments in which they remove the tubulin tails and tie the data into their final model. Nevertheless, there are several issues that require attention in order for the manuscript to be suitable for publication in eLife.

Major experimental issues that need to be addressed:

There is still lingering concern about the role of the C-terminal domain in the catalytic cycle of MCAK.

1) The authors show that the MCAK motor domain binds to microtubules with good binding affinity and that the modified (Cys containing) C-terminal peptide does not affect the binding affinity under reducing conditions. They have already clarified that the Cys-containing C-terminal peptide does not bind MCAK under reducing conditions, i.e. it does not trigger dimerization of MCAK. So the binding affinity for MTs they are observing now is the binding affinity of the monomeric motor domain for MTs. Next, the authors remove the reducing agent and observe that the fraction of MCAK bound to MTs has decreased. They claim that this is the expected behavior and illustrate the result in Figure 5C by showing that two equimolar populations of MCAK form, one of which can bind MTs and one of which cannot. This expectation is not well justified. How much of each of these two populations is represented depends on the concentration of peptide and on the time the peptide is given to form a covalent bond with MCAK, so that telling what is the expected result for this experiment is very hard unless all the reaction parameters are at hand. If it is true that the MCAK motor domain dimer:peptide has no or lower binding affinity for MT, peptide addition should result in progressively lower MCAK binding as a function of the concentration of C-terminal peptide and the time it is allowed to interact with MCAK.

The problem is that this experiment is a “remnant” from the theory that binding of the C-terminal tail to the motor domain is part of the catalytic cycle of MCAK and from the authors' attempts to lock this putative intermediate. This theory is downplayed in the present version and the authors could try to do the experiment in a simpler and more convincing setting. They don't need a covalent linker. They have created a full length MCAK with the S715E mutant, which they claim prevents binding of the C-terminal peptide to the motor domain. They can simply measure the binding affinity of this construct, which is because another segment of MCAK makes it be dimeric (SEC-MALS analysis in the subsection headed “The C terminus stabilizes full-length MCAK in solution”), in the presence of growing concentrations of free C-terminal peptide. As there should be no intra-molecular competition from the C-terminal tail (due to the S715E mutation) this experiment will clarify the actual effects of the C-terminal peptide in the absence of other confounding effects, particularly the underlying monomer-dimer equilibrium of the MCAK motor domain.

2) Concerning Figure 5: The authors need to do a control of MD no DTT, to show that the cross linker (rather than just lack of DTT) is causing the effects.

Other issues:

3) The manuscript suffers from a number of minor mistakes which would need to be corrected (listed below). It also could be improved by a clearer and more concise writing style. Parts of it are really hard to understand without reading it multiple times. In particular, the discussion of the experiments in Figure 5 is really cursory: the inferences are stated and the reader is simply referred to the figure, for which the figure legend provides minimal information. This is a key figure describing crucial experimental data, and the authors should explain the experiments, the resulting data and inferences completely. They may choose to break the figure up into multiple panels to achieve this end.

4) Figure 1: In the legend to Figure 1B the authors write: “The star represents residual GST”. Only later in the manuscript did this become clear. They need to explain that CT was made as a GST fusion and cleaved off. In Figure 1C and Figure 1D the order of the constructs is different. This is sloppy. The authors need to correct it.

5) Figure 1–figure supplement 1: this figure looks suspicious. The intensity of the peptide bands in the M+CT peak are very high relative to the CT bands. If they are staining equally it suggests a ratio of 20 peptides to 1 motor domain. Also there is a sharp drop off of intensity of the peptide band between fraction 10-11 and also 15-16. This does not look like a gel filtration peak. The authors need to address and possibly redo this gel. The fraction numbers on the gel need marking on the trace (which currently shows only volume).

6) Figure 3: A number of structural elements are referred to in the text that need to be shown on this figure: e.g. α1 and β3 of the dimerization interface, L1 loop, the interaction between Glu172 and Ala241/A is missing, Lys286/B is missing, structural elements of the ATPase site (P-loop, switch I and II). It would also be helpful to add an additional overlay structure to the figure that shows that these elements don't change upon C-tail binding.

7) Figure 4: A quadruple mutant (E711A/E712A/R716A/I718A) is shown in the figure but not discussed in the text. It should be removed or talked about. If it is kept then the way it is written above the gel needs to be improved as currently it is not clear that all 4 mutations were made in the same construct.

8) Figure 5: Figure 5E. The color lines in this figure are mislabelled. The magenta line should be MD+CTsc no DTT (rather than MD). In the figure legend there is reference to 6C, which should be 5D. The figure makes little sense otherwise. In this, and other figures, the axes should be more clearly explained in the figure legend. References in the text to the figures should also be readily understood by the reader. What is “Bmax”, referred to in the text but not apparent in the figure?

9) Figure 6: The cell images are too small and bad quality. They need to be made brighter and larger in order to show what is happening.

10) Figure 7: This figure is not currently mentioned in the text and needs to be.

11) In the first paragraph of the Results section, the term “upstream” is ambiguous. Consider using N or C terminal. The manuscript would be clearer if the authors maintained a consistent way of referring to the parts of the model. Sometimes they use CT and others “C-terminal domain” or “CT domain” or “C terminus”.

12) The authors should spell out clearly how they prepared CT alone (with no GST) and then clearly identify when they are using GST-CT and when CT.

13) In the second paragraph of the subsection “A conserved motif in the C-terminal region of MCAK is essential for the C terminus-motor interaction”, the term “head group” is confusing. The authors should state more clearly that they are changing the size of the amino acid at position 715.

14) In the subsection entitled “The C terminus-motor domain interaction is intramolecular”, the authors explain that removing the C-terminus of MCAK had no effect in depolymerisation assays. They then state “It is possible that there is a trade-off between microtubule binding affinity and tubulin removal at ends”. This sentence needs to be expanded to make it clearer. As I understand it they think there is no change in depolymerisation activity because of two counteracting effects (increase in activity and decrease in accessibility to the MT ends due to too much lattice binding). The authors could also consider moving this lack of change in activity to the discussion. It made reading the paper difficult.

15) The whole section on the intramolecular interaction is very difficult to follow and needs rephrasing to make it clearer. The authors may consider removing it as it does not seem to add to the overall argument.

16) In the second paragraph of the Results section: It may be easier to discuss the quantification of the binding affinity of the C-terminal peptide for the MCAK motor domain after describing the observation that the peptide causes dimerization of the C-terminal domain. The authors write: “…this measurement does not take into account any existing equilibrium between the motor domains (Figure 2B)”. In fact, the measurement results from the sum of two distinct interactions, the dimerization of the MCAK motor domain and the binding of the C-terminal peptide, an intrinsically cooperative interaction. It would be easier to clarify this if the dimerization had been already introduced.

17) At the end of the subsection headed “One MCAK C terminus binds to two ATPase domains”: “…we could not conclusively determine whether… there were additional dimerization domains in the context of full length MCAK”. The authors demonstrate later in the manuscript that MCAK forms dimers independently of the C-terminal domain, so this sentence may be confusing.

18) In the subsection “The MCAK C terminus binds at the interface between two motor domains”: There are four MCAK motor domains in the asymmetric unit of the crystals, two of which we are told are bound as a near-symmetrical dimer to a single peptide. The other two are not described, except for the explanation that they are not bound to the peptide because of a crystal contact. Are the two additional subunits that are not bound to the peptide in a dimeric arrangement? And also: If one considers only the two MCAK subunits bound to the peptide, are the two motor subunits related by a perfect (non-crystallographic) 2-fold axis? Clearly the presence of the peptide breaks the symmetry, but limitedly to the motor domain, are they in a perfect dimer? If so, it should be clearly stated.

19) At the end of the first paragraph of “The C terminus-motor domain interaction is intramolecular”: “However it did raise the possibility that the CT domain acts indirectly as an inhibitor and has an additional distinct cellular function”. “It” is a series of negative results discussed in the previous lines and showing that a point mutant that prevents binding of the CT has no effect in vitro and in vivo on microtubule depolymerization. It seems to me that emphasis here should be given to the negative results (some of which are not shown) rather than on the authors' goal of proving the importance of their finding.

20) In the second paragraph of the subheading “The C terminus-motor domain interaction is intramolecular”, the authors hypothesize that the reason why they do not see a covalent dimer form under oxidizing conditions with full length MCAK E712C is that the linkage is intra-molecular. It is possible, but the alternative explanation, that the linkage is not formed at all in the context of the full-length protein, is more plausible given the considerable number of residues lying in between the motor domain and the C-terminus.

21) At the end of the subsection headed “A conserved motif in the C-terminal region of MCAK is essential for the C terminus-motor interaction”: “data” is plural.

22) In the beginning of the section entitled “The C terminus-motor domain interaction is intramolecular”, the sentence starting with “It is possible that there is a trade-off…” is obscure.

23) In the subsection headed “The C terminus stabilizes full-length MCAK in solution”, it is not clear what is the evidence that MCAK S715S is less stable. If this referred to the stability of the dimer, it would be useful that the authors clarified how they reached this conclusion. As it stands, the inference that this mutant is less stable should be removed, as it is not justified.

Major experimental issues that need to be addressed:

There is still lingering concern about the role of the C-terminal domain in the catalytic cycle of MCAK.

1) The authors show that the MCAK motor domain binds to microtubules with good binding affinity and that the modified (Cys containing) C-terminal peptide does not affect the binding affinity under reducing conditions. They have already clarified that the Cys-containing C-terminal peptide does not bind MCAK under reducing conditions, i.e. it does not trigger dimerization of MCAK. So the binding affinity for MTs they are observing now is the binding affinity of the monomeric motor domain for MTs. Next, the authors remove the reducing agent and observe that the fraction of MCAK bound to MTs has decreased. They claim that this is the expected behavior and illustrate the result in Figure 5C by showing that two equimolar populations of MCAK form, one of which can bind MTs and one of which cannot. This expectation is not well justified. How much of each of these two populations is represented depends on the concentration of peptide and on the time the peptide is given to form a covalent bond with MCAK, so that telling what is the expected result for this experiment is very hard unless all the reaction parameters are at hand. If it is true that the MCAK motor domain dimer:peptide has no or lower binding affinity for MT, peptide addition should result in progressively lower MCAK binding as a function of the concentration of C-terminal peptide and the time it is allowed to interact with MCAK.

The problem is that this experiment is a “remnant” from the theory that binding of the C-terminal tail to the motor domain is part of the catalytic cycle of MCAK and from the authors' attempts to lock this putative intermediate. This theory is downplayed in the present version and the authors could try to do the experiment in a simpler and more convincing setting. They don't need a covalent linker. They have created a full length MCAK with the S715E mutant, which they claim prevents binding of the C-terminal peptide to the motor domain. They can simply measure the binding affinity of this construct, which is because another segment of MCAK makes it be dimeric (SEC-MALS analysis in the subsection headed “The C terminus stabilizes full-length MCAK in solution”), in the presence of growing concentrations of free C-terminal peptide. As there should be no intra-molecular competition from the C-terminal tail (due to the S715E mutation) this experiment will clarify the actual effects of the C-terminal peptide in the absence of other confounding effects, particularly the underlying monomer-dimer equilibrium of the MCAK motor domain.

We have now performed this experiment. When we titrate increasing amounts of free CT into the MCAK_S715E-microtubule reaction, we gradually decreased the amount of binding to the microtubule, revealing that the CT domain indeed reduces the affinity of MCAK for microtubules.

2) Concerning Figure 5: The authors need to do a control of MD no DTT, to show that the cross linker (rather than just lack of DTT) is causing the effects.

We have performed these experiments in Figure 6–figure supplement 3, where the motor domain (M) and the CT domain (CT) were incubated in absence of DTT. We then performed cosedimentation and microtubule depolymerization assays in presence of M+CT without reducing agent and we did not observe any changes in MCAK activity. Therefore these data indicate that it is specifically the covalent linkage between the motor and the CT_E712C in absence of DTT that affects MCAK function.

Other issues:

3) The manuscript suffers from a number of minor mistakes which would need to be corrected (listed below). It also could be improved by a clearer and more concise writing style. Parts of it are really hard to understand without reading it multiple times. In particular, the discussion of the experiments in Figure 5 is really cursory: the inferences are stated and the reader is simply referred to the figure, for which the figure legend provides minimal information. This is a key figure describing crucial experimental data, and the authors should explain the experiments, the resulting data and inferences completely. They may choose to break the figure up into multiple panels to achieve this end.

We have now broken up Figure 5 into two additional panels: one of them includes the experiment to major point 1, raised by the reviewers. We have also modified the text and figure legends to explain the experiments better.

4) Figure 1: In the legend to Figure 1B the authors write: “The star represents residual GST”. Only later in the manuscript did this become clear. They need to explain that CT was made as a GST fusion and cleaved off. In Figure 1C and Figure 1D the order of the constructs is different. This is sloppy. The authors need to correct it.

We have now stated that the tail domain (CT) is cleaved off from the GST and the GST is removed (in the first paragraph of the Results and in the Methods section). We used a smaller chemically synthesized peptide for complex crystallization and we have indicated this in the text, that the peptide was chemically synthesized (please see the subsection entitled “The MCAK C terminus binds at the interface between two motor domains”). We have also changed the order of constructs in Figure 1D.

5) Figure 1–figure supplement 1: this figure looks suspicious. The intensity of the peptide bands in the M+CT peak are very high relative to the CT bands. If they are staining equally it suggests a ratio of 20 peptides to 1 motor domain. Also there is a sharp drop off of intensity of the peptide band between fraction 10-11 and also 15-16. This does not look like a gel filtration peak. The authors need to address and possibly redo this gel. The fraction numbers on the gel need marking on the trace (which currently shows only volume).

We apologize for this problem and we agree it was imperative to change the figure. We have now repeated the gel filtration of the M, M+CT and M+CT_mutants constructs on appropriate gels (Invitrogen 16% Tricine gels) and have displayed the corresponding gels and elution profiles in Figures 1 and 4. We have removed the gel filtration of the M+GST-CT constructs for clarity.

6) Figure 3: A number of structural elements are referred to in the text that need to be shown on this figure: e.g. α1 and β3 of the dimerization interface, L1 loop, the interaction between Glu172 and Ala241/A is missing, Lys286/B is missing, structural elements of the ATPase site (P-loop, switch I and II). It would also be helpful to add an additional overlay structure to the figure that shows that these elements don't change upon C-tail binding.

We thank the reviewers for these excellent suggestions. They really help to understand the functional aspects of our MCAK structure better. We have now included new figures (Figure 3–figure supplement 1) with overlays of the mouse MCAK structure published by the Hirokawa group in 2004, with our MCAK structure. We then show that the ATPase site and the switch I and II regions are unperturbed while the neck linker region changes orientation around Lys258, with His257 interacting with the C-terminus. We have also included annotations to α1 and β3 in the dimerization interface and generated a figure that highlights the interaction between Lys286/B and Glu244/A, as it is only present when the CT domain is bound to the motor in our structure.

7) Figure 4: A quadruple mutant (E711A/E712A/R716A/I718A) is shown in the figure but not discussed in the text. It should be removed or talked about. If it is kept then the way it is written above the gel needs to be improved as currently it is not clear that all 4 mutations were made in the same construct.

We have now removed this from the figure and repeated the pulldown assay in Figure 4. At the same time, we have included the experiment in Figure 4–figure supplement 1A showing that the CT_S715A binds to the motor into Figure 4B.

8) Figure 5: Figure 5E. The color lines in this figure are mislabelled. The magenta line should be MD+CTsc no DTT (rather than MD). In the figure legend there is reference to 6C, which should be 5D. The figure makes little sense otherwise. In this, and other figures, the axes should be more clearly explained in the figure legend. References in the text to the figures should also be readily understood by the reader. What is “Bmax”, referred to in the text but not apparent in the figure?

We thank the reviewers and have now corrected this mislabeled figure. We have also relabeled the graph axes to explain more clearly what they represent and removed the reference to “Bmax” in the text. We have also edited the references to the figures.

9) Figure 6: The cell images are too small and bad quality. They need to be made brighter and larger in order to show what is happening.

We have now improved the presentation of the images to show the EB3 and MCAK staining more closely. We have also added linescan profile averages for the EB3 and MCAK localization on microtubule plus ends.

10) Figure 7: This figure is not currently mentioned in the text and needs to be.

We apologize for this mistake and have now mentioned Figure 7 (now Figure 8) in the text.

11) In the first paragraph of the Results section, the term “upstream” is ambiguous. Consider using N or C terminal. The manuscript would be clearer if the authors maintained a consistent way of referring to the parts of the model. Sometimes they use CT and others “C-terminal domain” or “CT domain” or “C terminus”.

We have now removed the term “upstream”. Throughout the text, we have more consistently labeled the C-terminus of MCAK construct as the CT domain. We apologize for this inconsistency.

12) The authors should spell out clearly how they prepared CT alone (with no GST) and then clearly identify when they are using GST-CT and when CT.

We have now described in the Methods section more clearly how the CT domain is prepared and how the GST is removed. We have also carefully annotated the figures and have tried to avoid using GST-CT when possible.

13) In the second paragraph of the subsection headed “A conserved motif in the C-terminal region of MCAK is essential for the C terminus-motor interaction”, the term “head group” is confusing. The authors should state more clearly that they are changing the size of the amino acid at position 715.

We have now changed the term “head group” to “side chain”.

14) In the subsection headed “The C terminus-motor domain interaction is intramolecular”, the authors explain that removing the C-terminus of MCAK had no effect in depolymerisation assays. They then state “It is possible that there is a trade-off between microtubule binding affinity and tubulin removal at ends”. This sentence needs to be expanded to make it clearer. As I understand it they think there is no change in depolymerisation activity because of two counteracting effects (increase in activity and decrease in accessibility to the MT ends due to too much lattice binding). The authors could also consider moving this lack of change in activity to the discussion. It made reading the paper difficult.

We have now changed this sentence to: “However there are limitations to this assay, in which we can only measure rate of microtubule depolymerization. It is possible that a change in MCAK microtubule binding affinity will have a counteracting effect on MCAK diffusion rate or the rate of tubulin removal at ends and that as a consequence. The overall depolymerase activity that our assay measures is unchanged, as previously shown (Cooper et al., 2010).”

15) The whole section on the intramolecular interaction is very difficult to follow and needs rephrasing to make it clearer. The authors may consider removing it as it does not seem to add to the overall argument.

We have now removed this section to streamline the paper.

16) In the second paragraph of the Results section: It may be easier to discuss the quantification of the binding affinity of the C-terminal peptide for the MCAK motor domain after describing the observation that the peptide causes dimerization of the C-terminal domain. The authors write: “…this measurement does not take into account any existing equilibrium between the motor domains (Figure 2B)”. In fact, the measurement results from the sum of two distinct interactions, the dimerization of the MCAK motor domain and the binding of the C-terminal peptide, an intrinsically cooperative interaction. It would be easier to clarify this if the dimerization had been already introduced.

We apologize for discussing motor dimerization before we introduced the data relative to it in the paper and we thank the reviewers for this comment. We have now moved the section quantifying the affinity of the peptide for the motor so that it follows the section describing that the CT domain induces motor dimerization. We have clarified that the affinity measured represents the sum of two distinct affinities, of which CT domain binding is cooperative.

17) At the end of the subsection headed “One MCAK C terminus binds to two ATPase domains”: “…we could not conclusively determine whether… there were additional dimerization domains in the context of full length MCAK”. The authors demonstrate later in the manuscript that MCAK forms dimers independently of the C-terminal domain, so this sentence may be confusing.

We have now included “from the above experiments” to highlight that the data described in that paragraph does not indicate whether MCAK is still dimeric or not.

18) In the subsection “The MCAK C terminus binds at the interface between two motor domains”: There are four MCAK motor domains in the asymmetric unit of the crystals, two of which we are told are bound as a near-symmetrical dimer to a single peptide. The other two are not described, except for the explanation that they are not bound to the peptide because of a crystal contact. Are the two additional subunits that are not bound to the peptide in a dimeric arrangement? And also: If one considers only the two MCAK subunits bound to the peptide, are the two motor subunits related by a perfect (non-crystallographic) 2-fold axis? Clearly the presence of the peptide breaks the symmetry, but limitedly to the motor domain, are they in a perfect dimer? If so, it should be clearly stated.

We thank the reviewers for this excellent point. We have now changed the text to reflect the structure better and have included this information and an additional figure of the CT binding site on the dimer that is not occupied by the CT domain.

19) At the end of the first paragraph of “The C terminus-motor domain interaction is intramolecular”: “However it did raise the possibility that the CT domain acts indirectly as an inhibitor and has an additional distinct cellular function”. “It” is a series of negative results discussed in the previous lines and showing that a point mutant that prevents binding of the CT has no effect in vitro and in vivo on microtubule depolymerization. It seems to me that emphasis here should be given to the negative results (some of which are not shown) rather than on the authors' goal of proving the importance of their finding.

We have measured the activity of MCAK and MCAK mutant such as MCAK_S175E using an assay first described by Moore and Wordeman (2004). The assay measures microtubule depolymerase activity in cells, by quantifying the microtubule fluorescence. However, while expression of MCAK depolymerizes microtubules, overexpression leads to microtubule bundling and abnormalities. We believe there are some limitations to this assay. Thus we could not measure with confidence significant differences in MCAK microtubule depolymerase activity upon removal of the CT domain and publish this data. We also used the PlusTipTracker (Danuser group) and a cell line expressing 2xGFP-EB3 to detect MCAK-dependent changes in microtubule-dynamics, by transiently transfecting MCAK and MCAK_S715E. However, the sample heterogeneity was in the order of magnitude of the changes recorded for the distinct mutants and transfection of GFP-MCAK rescued growth lifetime, but not growth speed. Therefore we did not include this data.

Finally, another paper (Hertzer et al., 2006) reported that removal of the CT domain resulted in a decrease in microtubule depolymerase activity, thus the effect of the CT domain on MCAK activity remains under debate. We have now included this information and the reference to the paper in this section.

20) In the second paragraph of the subheading “The C terminus-motor domain interaction is intramolecular”, the authors hypothesize that the reason why they do not see a covalent dimer form under oxidizing conditions with full length MCAK E712C is that the linkage is intra-molecular. It is possible, but the alternative explanation, that the linkage is not formed at all in the context of the full-length protein, is more plausible given the considerable number of residues lying in between the motor domain and the C-terminus.

We believe that we do form the crosslink because we see a reduction in the affinity of MCAK_E712C for microtubules in absence of DTT (Figure 5–figure supplement 1D). However, Cys287 (Cys283 in mouse) has also been reported to make a disulfide bond with Cys245 (Cys241 in mouse) in the murine MCAK crystal structure. Thus it is possible, that in the context of the full length MCAK and in absence of DTT, we generate at least two MCAK conformations of neck-motor complexes that make the analysis of this MCAK_E712C mutant more complex.

21) At the end of the subsection headed “A conserved motif in the C-terminal region of MCAK is essential for the C terminus-motor interaction”: “data” is plural.

We apologize and have now corrected this grammatical mistake.

22) In the beginning of the section entitled “The C terminus-motor domain interaction is intramolecular”, the sentence starting with “It is possible that there is a trade-off…” is obscure.

We have now clarified this sentence in the text.

23) In the subsection headed “The C terminus stabilizes full-length MCAK in solution”, it is not clear what is the evidence that MCAK S715S is less stable. If this referred to the stability of the dimer, it would be useful that the authors clarified how they reached this conclusion. As it stands, the inference that this mutant is less stable should be removed, as it is not justified.

The recovery rate from SEC-MALS was 33%, which implies some of the protein was not stable, but we did not include that data and we apologize for it. However, we have now removed the SEC-MALS data for MCAK_S715E and only included the gel filtration elution profile of MCAK_S715E.