Myristoyl’s dual role in allosterically regulating and localizing Abl kinase
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eLife assessment
This is an important study of the mechanism of how binding of the fatty acid myristic acid (MYR) inhibits the activity of the kinase c-Abl, a critical regulator of many cellular processes. While the general aspects of this regulation are known from structure determination and biochemical studies, the exact molecular mechanism and the nature of the allosteric inhibition were not known. The authors use MD simulation to close this gap and provide a detailed mechanistic description of the inhibitory mechanism, although some of the evidence remains incomplete.
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Abstract
c-Abl kinase, a key signaling hub in many biological processes ranging from cell development to proliferation, is tightly regulated by two inhibitory Src homology domains. An N-terminal myristoyl modification can bind to a hydrophobic pocket in the kinase C-lobe, which stabilizes the autoinhibitory assembly. Activation is triggered by myristoyl release. We used molecular dynamics simulations to show how both myristoyl and the Src homology domains are required to impose the full inhibitory effect on the kinase domain and reveal the allosteric transmission pathway at residue-level resolution. Importantly, we find myristoyl insertion into a membrane to thermodynamically compete with binding to c-Abl. Myristoyl thus not only localizes the protein to the cellular membrane, but membrane attachment at the same time enhances activation of c-Abl by stabilizing its preactivated state. Our data put forward a model in which lipidation tightly couples kinase localization and regulation, a scheme that currently appears to be unique for this non-receptor tyrosine kinase.
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Author Response
Reviewer #1 (Public Review):
The authors used MD simulations to investigate the role of N-terminal myristoylation and the presence of two SH domains on the allosteric regulation of c-Abl kinase. Standard established MD simulation methods and analyses were applied, including the force distribution analysis (FDA) method developed by Grater et al. some time ago.
The system is large and the conformational changes are complicated. In light of this, and aggravated by the fact that direct comparison with - and critical testing against - experimental data is not possible in the present case, I consider the overall simulation times to be rather short (several repeats, but only 500 ns). So there might be statistical convergence issues. Especially also because at least some of the starting structures were generated from …
Author Response
Reviewer #1 (Public Review):
The authors used MD simulations to investigate the role of N-terminal myristoylation and the presence of two SH domains on the allosteric regulation of c-Abl kinase. Standard established MD simulation methods and analyses were applied, including the force distribution analysis (FDA) method developed by Grater et al. some time ago.
The system is large and the conformational changes are complicated. In light of this, and aggravated by the fact that direct comparison with - and critical testing against - experimental data is not possible in the present case, I consider the overall simulation times to be rather short (several repeats, but only 500 ns). So there might be statistical convergence issues. Especially also because at least some of the starting structures were generated from available experimental structures after some modifications/modelling, and they might thus be out of equilibrium and need some time to fully relax during the MD simulations.
Unfortunately, I cannot find any convergence tests concerning the length of the simulations, which are usually considered to be standard analyses (Appendix Fig. 5 shows the effect of different thermostats and capping of the peptide chain, but no tests concerning simulation time). This could be critical in the present case, where the authors acknowledge themselves (e.g., on p. 4) that there are only subtle differences between the different simulation systems and the variations within a given system are larger than the relevant (putative) differences between systems (Fig. 1 C, D, E).
We thank the reviewer for taking the time and critically assessing our manuscript. We appreciate and have addressed the raised concerns as follows. We have quadrupled the simulation time to 2 µs for 20 out of the 30 replicates and show the updated results for these. We refer the reviewer to the modified Fig. 2 and 3 (former Fig. 1 and 2) with the updated data. Our main conclusions remained unchanged, namely that Myr unbinding shifts the overall kinase domain dynamics towards an active state. We furthermore still observe allosteric signal propagation from the Myr binding site to the active site along the alpha_F helix and a collaborative effect of Myr and the SH domains. Only some minor points were not confirmed after analyzing the longer simulations, for example the force differences transmitted to the A-loop upon SH domain binding/unbinding (former Fig. 2D), and changes in amplitude of N- and C-lobe opening upon Myr unbinding (former Fig. 1E). Furthermore, to demonstrate convergence, we added block and autocorrelation analyses for Fig. 1 (now Fig. 2) to Fig. 2 – fig supplement 3, and observed good convergence across all systems. Finally, we also increased simulation times of the umbrella sampling from 50ns to 200ns, again without that the quantitative trends and our conclusions have changed (see also next point).
Issues with statistical convergence are expected not only for the standard MD simulations but also for the umbrella sampling simulations, as 50 ns sampling per window is nowadays not considered state of the art and is likely insufficient for quantitative binding free energy calculation, especially for membranes (see, e.g., DOI 10.1021/ct200316w). However, worrying about this latter aspect might neither be useful nor needed, because in our view the statement that myristoyl groups can bind to the membrane and that they can compete with binding in the hydrophobic protein pocket can hardly be considered a surprise and would not have required any simulation at all in my view because the experimental K_D values are available (Table 1). The very unfavourable K_d values for unbinding of Myr from both the hydrophobic protein pocket as well as from the membrane in fact show that this is not how it is expected to work in reality. The fully solvated state will be avoided due to its high free energy. Instead, isn't the myristoyl expected to directly transition from the pocket into the membrane, after membrane binding of the kinase in a proper orientation?
The experimental values were determined with different methods, i.e. estimated from zeta potential measurements in case of the membrane and calorimetry, which only considered the kinase domain instead of the SH3-SH2-kinase complex, in case of Abl. We thus found it appropriate to perform Umbrella Sampling simulations to ensure comparability. Additionally, these allowed us to study the effects of different alpha_I helix conformations, which had a significant impact on the free energy of Myr unbinding, precisely Abl with a partially unfolded helix reflected the experimental energy better than the crystal structure with a kinked helix. We highlight this more explicitly in the corresponding Discussion section. Regarding the simulation time per sampling window, we did a block analysis (Fig. 5 – fig supplement 1) as suggested in the cited reference and also extended the time of each sampling window from 50 ns to 200 ns. This did not significantly alter the results and, importantly, the relative differences between Abl and the membrane stayed the same and are in good agreement with the experimental values.
Concerning the metadynamics simulations, these are usually done to obtain a free energy landscape. Why was this not attempted here? In the present case, the authors seemed to have used metadynamics only for generating starting structures, with different degrees of helicity of the alpha_I part, for subsequent standard MD simulations. Not surprisingly, nothing much happened during the latter, and conformers with kinked/partially unfolded alpha_I as well as conformers with straight alpha_I were both found to be "stable", at least on the short simulation time scale. It could also not be expected that the SH domain would spontaneously detach in response to helix straightening - again, this would require much longer simulation times than 500 ns. Nevertheless, alpha_I straightening might very well reduce the binding affinity towards SH - this can only be explicitly studied with free energy simulations, however.
Our main goal was indeed to achieve different alpha_I helix conformations for subsequent Umbrella Sampling simulations, and found that helix formation is in principle possible without SH2 domain unbinding. We would like to emphasize the impact of the different helix conformations on the free energy of Myr unbinding, which further highlights the need to investigate these structures. We chose Metadynamics to obtain them because it only facilitates the transition away from the kinked conformation without biasing towards certain end structures or transition pathways, which we found advantageous compared to alternative methods such as targeted MD. The reason for not reporting a free energy surface is that we considered the helicity of all seven residues making up the kink within a single CV, which smeared the energy landscape to the point that it is almost completely flattened. Furthermore, orthogonal CVs such as new interactions between the alpha_I helix with the SH2 domain or positional adjustments of the SH2 domain would have to be considered for a reliable quantitative result. We nevertheless observed transient SH2 domain unbinding during the applied time scale and added histograms to Fig. 4 – fig supplement 1 (former appendix Fig. 4) to make this more obvious.
Reviewer #2 (Public Review):
The manuscript aims at understanding how the fatty acid ligand MYR inhibits the activity of Abl kinase. Despite a wealth of structural and biochemical data, a key mechanistic understanding of how MYR binding could inactive Abl was missing.
The authors used equilibrium and enhanced molecular dynamics (MD) simulations to masterfully answer open questions left by extensive experimental data in the mechanistic understanding of this system. The authors took advantage of several state-of-the-art simulation techniques and carefully planned simulations to extract a coherent understanding from a wealth of experimental facts.
The manuscript convincingly identifies an allosteric regulation by MYR. Allostery is often a source of confusion and sometimes is used as a magic catch-it-all explanation for poorly understood phenomena. Here, the authors show very compelling evidence of the existence of an allosteric mechanism. Also, they identify the physical origin of the allosteric pathway, providing a clear mechanistic understanding at the residue-level resolution. This is an impressive achievement.
We thank the reviewer for appreciating our work and its significance for understanding Abl regulation.
By leaving a pocket in the protein, MYR enables the protein's activation. But MYR is a highly hydrophobic molecule surrounded by water. Where could it go rather than quickly binding back to the protein pocket? By asking this reasonable question, the authors propose an exciting mechanistic hypothesis. The physical proximity of Abl kinase to a cellular membrane could lead to a competition between the protein and the membrane for MYR, leading to a novel layer of regulation for this kinase. Free energy calculations performed by the authors show that this hypothesis is reasonable from the thermodynamic point of view.
From a broader perspective, this manuscript is an important contribution to the discussion of four outstanding topics. 1) myristoylation is an example of lipidation, a post-translational modification where an acyl chain is covalently linked to a protein. The role of post-translational modifications has been greatly underappreciated and investigated in the MD community. However, as all the work on Sars-Cov2 and this contribution show, post-translational modifications can be crucial to understanding function. Ignoring them could lead to severely biased results. 2) the debate on the nature of allostery is still on the rage. Some authors claim that looking for a residue-level mechanistic chain of events that explains the allosteric action does not make sense and that the only way of thinking about allostery is as a sudden global change of the conformational landscape. Here, the authors show that instead, it is possible and leads to an essential understanding. 3) The authors hypothesize a novel crosstalk between the Abl and cellular membranes mediated by MYR. This exciting and far-reaching hypothesis opens the door to new complex layers of regulation. I suspect that these crosstalks between cytosolic proteins, or the soluble domain of membrane-tethered proteins and membranes, are much more ubiquitous than what has been appreciated so far. 4) From a methodological point of view, this manuscript represents a masterful use of simulations to put existing experimental data in a coherent picture. It is an example of the use of MD simulations at its best, where the simulations make sense of experiments, integrate existing data into a unified picture, and lead to new hypotheses that can be tested in future experiments.
We thoroughly appreciate the reviewers positive feedback and the valuable suggestions for improvement below.
It would be superb if the authors could propose precise predictions that could inspire future experiments. Now that they present a residue-resolution allosteric pathway, can they suggest point mutations that would interrupt it?
We have added a short segment to the end of the discussion proposing possible experiments.
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eLife assessment
This is an important study of the mechanism of how binding of the fatty acid myristic acid (MYR) inhibits the activity of the kinase c-Abl, a critical regulator of many cellular processes. While the general aspects of this regulation are known from structure determination and biochemical studies, the exact molecular mechanism and the nature of the allosteric inhibition were not known. The authors use MD simulation to close this gap and provide a detailed mechanistic description of the inhibitory mechanism, although some of the evidence remains incomplete.
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Reviewer #1 (Public Review):
The authors used MD simulations to investigate the role of N-terminal myristoylation and the presence of two SH domains on the allosteric regulation of c-Abl kinase. Standard established MD simulation methods and analyses were applied, including the force distribution analysis (FDA) method developed by Grater et al. some time ago.
The system is large and the conformational changes are complicated. In light of this, and aggravated by the fact that direct comparison with - and critical testing against - experimental data is not possible in the present case, I consider the overall simulation times to be rather short (several repeats, but only 500 ns). So there might be statistical convergence issues. Especially also because at least some of the starting structures were generated from available experimental …
Reviewer #1 (Public Review):
The authors used MD simulations to investigate the role of N-terminal myristoylation and the presence of two SH domains on the allosteric regulation of c-Abl kinase. Standard established MD simulation methods and analyses were applied, including the force distribution analysis (FDA) method developed by Grater et al. some time ago.
The system is large and the conformational changes are complicated. In light of this, and aggravated by the fact that direct comparison with - and critical testing against - experimental data is not possible in the present case, I consider the overall simulation times to be rather short (several repeats, but only 500 ns). So there might be statistical convergence issues. Especially also because at least some of the starting structures were generated from available experimental structures after some modifications/modelling, and they might thus be out of equilibrium and need some time to fully relax during the MD simulations.
Unfortunately, I cannot find any convergence tests concerning the length of the simulations, which are usually considered to be standard analyses (Appendix Fig. 5 shows the effect of different thermostats and capping of the peptide chain, but no tests concerning simulation time). This could be critical in the present case, where the authors acknowledge themselves (e.g., on p. 4) that there are only subtle differences between the different simulation systems and the variations within a given system are larger than the relevant (putative) differences between systems (Fig. 1 C, D, E).
Issues with statistical convergence are expected not only for the standard MD simulations but also for the umbrella sampling simulations, as 50 ns sampling per window is nowadays not considered state of the art and is likely insufficient for quantitative binding free energy calculation, especially for membranes (see, e.g., DOI 10.1021/ct200316w). However, worrying about this latter aspect might neither be useful nor needed, because in our view the statement that myristoyl groups can bind to the membrane and that they can compete with binding in the hydrophobic protein pocket can hardly be considered a surprise and would not have required any simulation at all in my view because the experimental K_D values are available (Table 1). The very unfavourable K_d values for unbinding of Myr from both the hydrophobic protein pocket as well as from the membrane in fact show that this is not how it is expected to work in reality. The fully solvated state will be avoided due to its high free energy. Instead, isn't the myristoyl expected to directly transition from the pocket into the membrane, after membrane binding of the kinase in a proper orientation?
Concerning the metadynamics simulations, these are usually done to obtain a free energy landscape. Why was this not attempted here? In the present case, the authors seemed to have used metadynamics only for generating starting structures, with different degrees of helicity of the alpha_I part, for subsequent standard MD simulations. Not surprisingly, nothing much happened during the latter, and conformers with kinked/partially unfolded alpha_I as well as conformers with straight alpha_I were both found to be "stable", at least on the short simulation time scale. It could also not be expected that the SH domain would spontaneously detach in response to helix straightening - again, this would require much longer simulation times than 500 ns. Nevertheless, alpha_I straightening might very well reduce the binding affinity towards SH - this can only be explicitly studied with free energy simulations, however.
-
Reviewer #2 (Public Review):
The manuscript aims at understanding how the fatty acid ligand MYR inhibits the activity of Abl kinase. Despite a wealth of structural and biochemical data, a key mechanistic understanding of how MYR binding could inactive Abl was missing.
The authors used equilibrium and enhanced molecular dynamics (MD) simulations to masterfully answer open questions left by extensive experimental data in the mechanistic understanding of this system. The authors took advantage of several state-of-the-art simulation techniques and carefully planned simulations to extract a coherent understanding from a wealth of experimental facts.
The manuscript convincingly identifies an allosteric regulation by MYR. Allostery is often a source of confusion and sometimes is used as a magic catch-it-all explanation for poorly understood …
Reviewer #2 (Public Review):
The manuscript aims at understanding how the fatty acid ligand MYR inhibits the activity of Abl kinase. Despite a wealth of structural and biochemical data, a key mechanistic understanding of how MYR binding could inactive Abl was missing.
The authors used equilibrium and enhanced molecular dynamics (MD) simulations to masterfully answer open questions left by extensive experimental data in the mechanistic understanding of this system. The authors took advantage of several state-of-the-art simulation techniques and carefully planned simulations to extract a coherent understanding from a wealth of experimental facts.
The manuscript convincingly identifies an allosteric regulation by MYR. Allostery is often a source of confusion and sometimes is used as a magic catch-it-all explanation for poorly understood phenomena. Here, the authors show very compelling evidence of the existence of an allosteric mechanism. Also, they identify the physical origin of the allosteric pathway, providing a clear mechanistic understanding at the residue-level resolution. This is an impressive achievement.
By leaving a pocket in the protein, MYR enables the protein's activation. But MYR is a highly hydrophobic molecule surrounded by water. Where could it go rather than quickly binding back to the protein pocket? By asking this reasonable question, the authors propose an exciting mechanistic hypothesis. The physical proximity of Abl kinase to a cellular membrane could lead to a competition between the protein and the membrane for MYR, leading to a novel layer of regulation for this kinase. Free energy calculations performed by the authors show that this hypothesis is reasonable from the thermodynamic point of view.
From a broader perspective, this manuscript is an important contribution to the discussion of four outstanding topics. 1) myristoylation is an example of lipidation, a post-translational modification where an acyl chain is covalently linked to a protein. The role of post-translational modifications has been greatly underappreciated and investigated in the MD community. However, as all the work on Sars-Cov2 and this contribution show, post-translational modifications can be crucial to understanding function. Ignoring them could lead to severely biased results. 2) the debate on the nature of allostery is still on the rage. Some authors claim that looking for a residue-level mechanistic chain of events that explains the allosteric action does not make sense and that the only way of thinking about allostery is as a sudden global change of the conformational landscape. Here, the authors show that instead, it is possible and leads to an essential understanding. 3) The authors hypothesize a novel crosstalk between the Abl and cellular membranes mediated by MYR. This exciting and far-reaching hypothesis opens the door to new complex layers of regulation. I suspect that these crosstalks between cytosolic proteins, or the soluble domain of membrane-tethered proteins and membranes, are much more ubiquitous than what has been appreciated so far. 4) From a methodological point of view, this manuscript represents a masterful use of simulations to put existing experimental data in a coherent picture. It is an example of the use of MD simulations at its best, where the simulations make sense of experiments, integrate existing data into a unified picture, and lead to new hypotheses that can be tested in future experiments.
It would be superb if the authors could propose precise predictions that could inspire future experiments. Now that they present a residue-resolution allosteric pathway, can they suggest point mutations that would interrupt it?
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