The structural basis of the multi-step allosteric activation of Aurora B kinase
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eLife assessment
This important study investigates the dynamic activation mechanism of a key mitotic kinase complex, Aurora B/INCENP. The method of generating specifically phosphorylated forms of the complex is elegant, supporting a compelling biochemical analysis of how these sites synergistically activate Aurora B. However, the limitations of the molecular dynamics approach and how these models compare to previous structural studies are incompletely addressed. This work will be of interest to cell biologists and biochemists studying cell division and kinase regulation.
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Abstract
Aurora B, together with IN-box, the C-terminal part of INCENP, forms an enzymatic complex that ensures faithful cell division. The [Aurora B/IN-box] complex is activated by autophosphorylation in the Aurora B activation loop and in IN-box, but it is not clear how these phosphorylations activate the enzyme. We used a combination of experimental and computational studies to investigate the effects of phosphorylation on the molecular dynamics and structure of [Aurora B/IN-box]. In addition, we generated partially phosphorylated intermediates to analyze the contribution of each phosphorylation independently. We found that the dynamics of Aurora and IN-box are interconnected, and IN-box plays both positive and negative regulatory roles depending on the phosphorylation status of the enzyme complex. Phosphorylation in the activation loop of Aurora B occurs intramolecularly and prepares the enzyme complex for activation, but two phosphorylated sites are synergistically responsible for full enzyme activity.
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Author Response
Reviewer #1 (Public Review):
This is a well-conceived and well-executed investigation of how activation loop autophosphorylation and IN-box autophosphorylation synergistically activate AURKB/INCENP. An elegant chemical ligation strategy allowed construction of the intermediate phospho-forms so that the contributions of each phosphorylation event to structure, dynamics, and activity could be dissected. Autophosphorylation at both sites serves to rigidify both AURKB and the IN-box, and to coordinate opening, twisting, and activation loop movements. Consistent with previous findings, both sites are necessary for enzymatic activity; further, this work finds that activation loop autophosphorylation occurs slowly in cis while INbox autophosphorylation occurs quickly in trans.
Due to abundant previous work in the field, …
Author Response
Reviewer #1 (Public Review):
This is a well-conceived and well-executed investigation of how activation loop autophosphorylation and IN-box autophosphorylation synergistically activate AURKB/INCENP. An elegant chemical ligation strategy allowed construction of the intermediate phospho-forms so that the contributions of each phosphorylation event to structure, dynamics, and activity could be dissected. Autophosphorylation at both sites serves to rigidify both AURKB and the IN-box, and to coordinate opening, twisting, and activation loop movements. Consistent with previous findings, both sites are necessary for enzymatic activity; further, this work finds that activation loop autophosphorylation occurs slowly in cis while INbox autophosphorylation occurs quickly in trans.
Due to abundant previous work in the field, many of the conclusions of this paper were expected. However, that does not diminish the quality of the work, and the addition of how kinase dynamics contribute to activation is important for AURKB and many other kinases. The experimental results are clear and interpreted appropriately, with good controls. The computational work is also clearly explained and directly tied to the function of the enzyme, making it highly complementary to the experimental findings and to previously published structures.
We thank the reviewer for positive words about our work.
Some minor limitations of the study:
- Of note when interpreting the HDX data, there is no coverage of the peptide containing the activation loop autophosphorylation site T248 (Fig S2A), and as mentioned in the Discussion, the time scale of HDX is not able to capture differences in exchange in very flexible regions like the activation loop.
The peptides spanning the region containing the phosphorylated Aurora BThr248 are not shown in our coverage map because they do not meet the stringent quality criteria for peptides that we used for HDX analysis (see Material and Methods). However, we have compared these peptides in phosphorylated and unphosphorylated enzyme complex manually and added a paragraph 4 on page 4.
“The peptides spanning the region containing the phosphorylated Aurora BThr248 are not shown in our coverage map (Figure 1-figure supplement 2A) because they did not pass the stringent peptide quality filter based on intensity, and redundancy of the peptide. However, upon manual analysis, we did not detect any changes in deuterium uptake between the phosphorylated and unphosphorylated forms in this region. Deuterium exchange in this part of the protein (which is also observed in the peptides immediately upstream of Aurora BThr248, see Supplementary file 5) is very rapid, independently of enzyme phosphorylation, so that complete exchange occurs even at the earliest time points. This is in contrast to the second part of the activation loop, which includes the Aurora BaEF helix (labeled region 3 in Figure 1A; peptide 254-260 in Supplementary file 5), where we clearly see HDX protection upon phosphorylation. It is possible that phosphorylation causes dynamic changes on a very fast scale (seconds or faster) in the part of the protein encompassing Aurora BThr248, but we could not detect them due to the limitation of our approach, which operates on the scale of minutes.“
Also, we analyzed the peptides covering this region to confirm the extent of phosphorylation in the loop (Figure 3-figure supplement 2A).
- Some data lack robust statistical analysis, which would make the findings more compelling.
We have now included statistical analysis throughout the paper where it was possible.
- One point that might be clarified is how the occupancy of T248 was confirmed to be either fully phosphorylated in the [AURKB/IN-box]IN-deltaC or fully dephosphorylated in the IN-box K846N/R827Q mutant. Especially because T248 autophosphorylation is found to occur in cis, it is unclear how incubating the [AURKB/IN-box]IN-deltaC with traces of wild-type [AURKB/IN-box]all-P would ensure that T248 is phosphorylated.
We confirmed phosphorylation occupancy of Aurora BThr248 by mass spectrometry in [Aurora B/IN-box]allP and [Aurora B/IN-box]loop-P but not for [AURKB/IN-box]no-P (Figure 3-figure supplement 2A). To achieve complete phosphorylation in the activation loop of [Aurora B/IN-box]IN-DC, we incubated this construct with fully active [Aurora B/IN-box]all-P. This is because, according to previous kinetic analysis, cisphosphorylation of Aurora BThr248 is only obligatory when the entire enzyme population is in the nonphosphorylated state. Once a fraction of the Aurora B population has been partially or fully activated, phosphorylation of Aurora BThr248 can also occur in trans, by the already activated enzyme. In other words, our model proposes an obligatory initial intramolecular step followed by propagation of activation in trans as reported by (Zaytsev, Segura-Peña at al, eLife.2016). We have now clarified this on page 11, paragraph 2 where we explain the results of the autoactivation kinetics.
“It is noteworthy that phosphorylation of the activation loop in cis is the first necessary step in the autoactivation process, assuming a completely unphosphorylated enzyme pool. However, a partially active or fully active enzyme can phosphorylate the activation loop in trans. This type of activation mechanism with an initial intramolecular activation step followed by an intermolecular step of activation have been previously reported for PAK2 (J. Wang et al., 2011) and for Aurora B (Zaytsev et al., 2016).”
Reviewer #2 (Public Review):
This study presents a dynamic, multi-step model for the activation of Aurora-B kinase through the interaction with INCENP and autophosphorylation. This interaction is critical to the proper execution of chromosome segregation, and key details of the mechanism are not resolved. The study is an advance on previous studies on Aurora-B and the related kinase Aurora-C, primarily because it clarifies the roles of the different phosphorylation sites. However, major differences in the details of the molecular interactions are presented that are not clearly backed up by the evidence due to limitations in the approach, when compared to previous work based on crystal structures.
Strengths. The experimental approach to the analysis of the Aurora-B/INCENP interaction is sound and novel and it is striking example of preparation of proteins in specific phosphorylation states, and of using HDX to characterise localised changes in the structural dynamics of a protein complex. The authors have generated two intermediate phosphorylation states of the complex, enabling them to dissect their contributions to the regulation of structural dynamics and activity of the complex.
Weaknesses. The major weakness of the study is the molecular dynamics simulation. The resulting model of the complex differs from the crystal structure of the Aurora-C/IN-box structure in key details, and these are neither described clearly nor explained. The challenges/limitations of simulation of phosphorylated proteins should be described.
We thank the reviewer for the positive words about our work and the criticism that helped us to improve our manuscript. We have now extended the MD studies that confirm our original observations regarding the entropic nature of the IN-box and the effect of phosphorylation on the structure and dynamics of [Aurora B/IN-box]. We clarify that the conformation of [Aurora B/IN-box]all-P observed in the simulations is not the final folded state, but a productive intermediate in the activation pathway of [Aurora B/IN-box]. For this reason, the differences from the [Aurora C/IN-box] crystal structure do not indicate flaws in the simulations. On the contrary, we believe that the data from the MD simulations provide crucial insights into the dynamical properties of the system that could not otherwise be assessed.
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eLife assessment
This important study investigates the dynamic activation mechanism of a key mitotic kinase complex, Aurora B/INCENP. The method of generating specifically phosphorylated forms of the complex is elegant, supporting a compelling biochemical analysis of how these sites synergistically activate Aurora B. However, the limitations of the molecular dynamics approach and how these models compare to previous structural studies are incompletely addressed. This work will be of interest to cell biologists and biochemists studying cell division and kinase regulation.
-
Reviewer #1 (Public Review):
This is a well-conceived and well-executed investigation of how activation loop autophosphorylation and IN-box autophosphorylation synergistically activate AURKB/INCENP. An elegant chemical ligation strategy allowed construction of the intermediate phospho-forms so that the contributions of each phosphorylation event to structure, dynamics, and activity could be dissected. Autophosphorylation at both sites serves to rigidify both AURKB and the IN-box, and to coordinate opening, twisting, and activation loop movements. Consistent with previous findings, both sites are necessary for enzymatic activity; further, this work finds that activation loop autophosphorylation occurs slowly in cis while IN-box autophosphorylation occurs quickly in trans.
Due to abundant previous work in the field, many of the …
Reviewer #1 (Public Review):
This is a well-conceived and well-executed investigation of how activation loop autophosphorylation and IN-box autophosphorylation synergistically activate AURKB/INCENP. An elegant chemical ligation strategy allowed construction of the intermediate phospho-forms so that the contributions of each phosphorylation event to structure, dynamics, and activity could be dissected. Autophosphorylation at both sites serves to rigidify both AURKB and the IN-box, and to coordinate opening, twisting, and activation loop movements. Consistent with previous findings, both sites are necessary for enzymatic activity; further, this work finds that activation loop autophosphorylation occurs slowly in cis while IN-box autophosphorylation occurs quickly in trans.
Due to abundant previous work in the field, many of the conclusions of this paper were expected. However, that does not diminish the quality of the work, and the addition of how kinase dynamics contribute to activation is important for AURKB and many other kinases. The experimental results are clear and interpreted appropriately, with good controls. The computational work is also clearly explained and directly tied to the function of the enzyme, making it highly complementary to the experimental findings and to previously published structures.
Some minor limitations of the study:
1. Of note when interpreting the HDX data, there is no coverage of the peptide containing the activation loop autophosphorylation site T248 (Fig S2A), and as mentioned in the Discussion, the time scale of HDX is not able to capture differences in exchange in very flexible regions like the activation loop.
2. Some data lack robust statistical analysis, which would make the findings more compelling.
3. One point that might be clarified is how the occupancy of T248 was confirmed to be either fully phosphorylated in the [AURKB/IN-box]IN-deltaC or fully dephosphorylated in the IN-box K846N/R827Q mutant. Especially because T248 autophosphorylation is found to occur in cis, it is unclear how incubating the [AURKB/IN-box]IN-deltaC with traces of wild-type [AURKB/IN-box]all-P would ensure that T248 is phosphorylated.
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Reviewer #2 (Public Review):
This study presents a dynamic, multi-step model for the activation of Aurora-B kinase through the interaction with INCENP and autophosphorylation. This interaction is critical to the proper execution of chromosome segregation, and key details of the mechanism are not resolved. The study is an advance on previous studies on Aurora-B and the related kinase Aurora-C, primarily because it clarifies the roles of the different phosphorylation sites. However, major differences in the details of the molecular interactions are presented that are not clearly backed up by the evidence due to limitations in the approach, when compared to previous work based on crystal structures.
Strengths. The experimental approach to the analysis of the Aurora-B/INCENP interaction is sound and novel and it is striking example of …
Reviewer #2 (Public Review):
This study presents a dynamic, multi-step model for the activation of Aurora-B kinase through the interaction with INCENP and autophosphorylation. This interaction is critical to the proper execution of chromosome segregation, and key details of the mechanism are not resolved. The study is an advance on previous studies on Aurora-B and the related kinase Aurora-C, primarily because it clarifies the roles of the different phosphorylation sites. However, major differences in the details of the molecular interactions are presented that are not clearly backed up by the evidence due to limitations in the approach, when compared to previous work based on crystal structures.
Strengths. The experimental approach to the analysis of the Aurora-B/INCENP interaction is sound and novel and it is striking example of preparation of proteins in specific phosphorylation states, and of using HDX to characterise localised changes in the structural dynamics of a protein complex. The authors have generated two intermediate phosphorylation states of the complex, enabling them to dissect their contributions to the regulation of structural dynamics and activity of the complex.
Weaknesses. The major weakness of the study is the molecular dynamics simulation. The resulting model of the complex differs from the crystal structure of the Aurora-C/IN-box structure in key details, and these are neither described clearly nor explained. The challenges/limitations of simulation of phosphorylated proteins should be described.
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Reviewer #3 (Public Review):
The chromosomal passenger complex (CPC) is an important regulator of mitotic progression, e.g. controlling kinetochore-microtubule attachment and cytokinesis. In this manuscript, Segura-Peña and colleagues investigated how the enzymatic core complex of the CPC, Aurora B and IN-box (the C-terminal part of INCENP), is structurally and functionally regulated by multiple (auto)phosphorylations. By doing so they are providing an insightful, dynamic picture of how the coordinated phosphorylations of the Aurora B T-loop and two serines in IN-box act cooperatively in order to fully activate the kinase.
Previously, several structures of Aurora B/IN-Box (missing the C-terminus of IN-box with two important phosphorylation sites or being unstructured, Sessa et al. 2005, Sessa and Villa et al. 2015, Elkins et al., 2012) …
Reviewer #3 (Public Review):
The chromosomal passenger complex (CPC) is an important regulator of mitotic progression, e.g. controlling kinetochore-microtubule attachment and cytokinesis. In this manuscript, Segura-Peña and colleagues investigated how the enzymatic core complex of the CPC, Aurora B and IN-box (the C-terminal part of INCENP), is structurally and functionally regulated by multiple (auto)phosphorylations. By doing so they are providing an insightful, dynamic picture of how the coordinated phosphorylations of the Aurora B T-loop and two serines in IN-box act cooperatively in order to fully activate the kinase.
Previously, several structures of Aurora B/IN-Box (missing the C-terminus of IN-box with two important phosphorylation sites or being unstructured, Sessa et al. 2005, Sessa and Villa et al. 2015, Elkins et al., 2012) and phosphorylated Aurora C/IN-Box (Abdul Azeez et al., 2019) had provided numerous structural insights and highlighted the role of the phosphorylated residues in T-loop and IN-box. Here, the authors now reveal the dynamic dimension of how the activity of this complex is regulated by using a compelling combination of H/D exchange mass spectrometry (HDX), molecular dynamics simulation and elegant biochemistry. Using HDX they demonstrate that upon Aurora B/IN-box autophosphorylation several regions of the complex become more structured. Using molecular dynamics, they explore the different conformational states of the complex and in particular how the phosphorylation and interactions of the phosphorylated C-terminal tail of IN-box coordinates and rigidifies Aurora B. To dissect the contributions of the phosphorylations on T-loop and IN-box, the authors create differentially phosphorylated versions of the complex using a sophisticated, intein-based protein engineering approach. The biochemical assays performed with these versions reveal not only the synergistic nature of these phosphorylation sites but also establish the nature of the autophosphorylation (cis for Aurora B, trans for IN-box) and show that Aurora B autophosphorylation in cis is rate-limiting. The data is convincing and intriguing, and remaining criticisms have been addressed extensively during the rewriting of the manuscript. In my opinion no additional experiments are required.
In summary, this is a well-executed study that provides new detailed molecular insights into the regulation of an important cell cycle complex. The findings and approaches will be of great interest to both the kinase and the cell cycle community.
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