Evolutionary divergence in the conformational landscapes of tyrosine vs serine/threonine kinases
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eLife assessment
This important paper provides a convincing mechanism for relative binding specificity of Type II inhibitors to kinases. The combination of a sequence-derived Potts-model with experimental dissociation constants and calculated free energies of binding to the DFG-out state is highly compelling and goes beyond the current state of the art. Given the importance of kinases in pathophysiological processes, the results will be of interest to a broad audience and, in addition, the combination of computational methods can be applicable to a wide variety of other biophysical processes that involve conformational rearrangements.
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Abstract
Inactive conformations of protein kinase catalytic domains where the DFG motif has a “DFG-out” orientation and the activation loop is folded present a druggable binding pocket that is targeted by FDA-approved ‘type-II inhibitors’ in the treatment of cancers. Tyrosine kinases (TKs) typically show strong binding affinity with a wide spectrum of type-II inhibitors while serine/threonine kinases (STKs) usually bind more weakly which we suggest here is due to differences in the folded to extended conformational equilibrium of the activation loop between TKs vs. STKs. To investigate this, we use sequence covariation analysis with a Potts Hamiltonian statistical energy model to guide absolute binding free-energy molecular dynamics simulations of 74 protein-ligand complexes. Using the calculated binding free energies together with experimental values, we estimated free-energy costs for the large-scale (~17–20 Å) conformational change of the activation loop by an indirect approach, circumventing the very challenging problem of simulating the conformational change directly. We also used the Potts statistical potential to thread large sequence ensembles over active and inactive kinase states. The structure-based and sequence-based analyses are consistent; together they suggest TKs evolved to have free-energy penalties for the classical ‘folded activation loop’ DFG-out conformation relative to the active conformation, that is, on average, 4–6 kcal/mol smaller than the corresponding values for STKs. Potts statistical energy analysis suggests a molecular basis for this observation, wherein the activation loops of TKs are more weakly ‘anchored’ against the catalytic loop motif in the active conformation and form more stable substrate-mimicking interactions in the inactive conformation. These results provide insights into the molecular basis for the divergent functional properties of TKs and STKs, and have pharmacological implications for the target selectivity of type-II inhibitors.
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eLife assessment
This important paper provides a convincing mechanism for relative binding specificity of Type II inhibitors to kinases. The combination of a sequence-derived Potts-model with experimental dissociation constants and calculated free energies of binding to the DFG-out state is highly compelling and goes beyond the current state of the art. Given the importance of kinases in pathophysiological processes, the results will be of interest to a broad audience and, in addition, the combination of computational methods can be applicable to a wide variety of other biophysical processes that involve conformational rearrangements.
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Reviewer #1 (Public Review):
The authors had previously developed a method of determining conformational free energy differences between the alternative DFG-in and DFG-out conformational states of kinases using an energy function based on a Potts model. They did this because direct estimates of this free energy change from molecular simulations, while possible in principle, would in practice be hard to do with sufficient accuracy to be useful for such a large conformational transition. Potts model energies have been shown to be correlated with overall protein stability, so it is reasonable that dividing the contacts into DFG-in and DFG-out sets should allow the estimation of a free energy difference between conformational states. In this work they examine the differences between Tyrosine Kinases (TKs) and Serine/Threonine Kinases (STKs) …
Reviewer #1 (Public Review):
The authors had previously developed a method of determining conformational free energy differences between the alternative DFG-in and DFG-out conformational states of kinases using an energy function based on a Potts model. They did this because direct estimates of this free energy change from molecular simulations, while possible in principle, would in practice be hard to do with sufficient accuracy to be useful for such a large conformational transition. Potts model energies have been shown to be correlated with overall protein stability, so it is reasonable that dividing the contacts into DFG-in and DFG-out sets should allow the estimation of a free energy difference between conformational states. In this work they examine the differences between Tyrosine Kinases (TKs) and Serine/Threonine Kinases (STKs) more closely, finding that the model predicts a small free energy change for converting DFG-in to DFG-out for TKs but a significant unfavorable free energy cost to converting to DFG-out for the STKs. The most insightful part of the paper comes in its analysis of how this conformational change may contribute to the overall binding free energies. Calculating binding free energies for Type II inhibitors (which bind DFG-out) by alchemical methods neglects the contribution from any unfavorable conformational change ("reorganization energy") required to adopt the DFG-out conformation. Thus comparing this calculated binding free energy with the total binding free energy estimated from experiment allows an estimate of the conformational reorganization energy. It is found that this estimate is nicely correlated with the free energy change for conformational rearrangement estimated from the Potts model analysis. Thus an important contribution to Type II inhibitor binding is this conformational transition. The different contributions to Type II binding are analyzed in detail by further dissecting the Potts model.
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Reviewer #2 (Public Review):
This paper focuses on an important topic. It explores how the activation loop conformations affect the type II inhibitor binding in Tyr and Ser/Thr kinases. The comprehensive computational results agree with the available experimental data. It is a remarkably comprehensive, high quality paper.
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Reviewer #3 (Public Review):
Tyrosine kinases (TKs) belong to a relatively small family of protein kinases that are a product of later evolution and play a critical role in the regulation of cell behavior in multicellular organisms. Major differences between TKs and Serine/threonine kinases (STKs) are very well known, however, it is still unclear if there are specific sequence signatures that favor a specific inactive conformation of TKs that can be exploited for efficient drug design. The authors used Potts Hamiltonian models (PHMs) along with other computational methods to tackle this problem. The are two main weaknesses of this approach. First, it relies on multiple sequence alignment that requires a large set of related sequences and can't be applied to smaller families. Second, it requires a relatively large number of structures …
Reviewer #3 (Public Review):
Tyrosine kinases (TKs) belong to a relatively small family of protein kinases that are a product of later evolution and play a critical role in the regulation of cell behavior in multicellular organisms. Major differences between TKs and Serine/threonine kinases (STKs) are very well known, however, it is still unclear if there are specific sequence signatures that favor a specific inactive conformation of TKs that can be exploited for efficient drug design. The authors used Potts Hamiltonian models (PHMs) along with other computational methods to tackle this problem. The are two main weaknesses of this approach. First, it relies on multiple sequence alignment that requires a large set of related sequences and can't be applied to smaller families. Second, it requires a relatively large number of structures that have similar inactive structures. Although all active kinases have very similar structures, their inactive structures are very diverse. However, there are several groups of inactive conformations that share a high level of similarity. The authors study one of them, the so-called "DFG-out" conformation, and present a set of convincing results that define several key residues that favor this conformation. They demonstrated the strength of the PHMs approach that allows the detection of critical contacts that are specific for certain conformations. These results can be used to predict the "DFG-out" conformation of a TK even if its structure is not known or predict the effects of mutations in a TK if they involve some of the critical residues. In general, the paper presents a set of solid results that will facilitate the development of highly specific inhibitors for TKs.
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