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How Electrostatic Coupling Enables Conformational Plasticity in a Tyrosine Kinase.

ABSTRACT: Protein kinases are important cellular signaling molecules involved in cancer and a multitude of other diseases. It is well-known that inactive kinases display a remarkable conformational plasticity; however, the molecular mechanisms remain poorly understood. Conformational heterogeneity presents an opportunity but also a challenge in kinase drug discovery. The ability to predictively model various conformational states could accelerate selective inhibitor design. Here we performed a proton-coupled molecular dynamics study to explore the conformational landscape of a c-Src kinase. Starting from a completely inactive structure, the simulations captured all major types of conformational states without the use of a target structure, mutation, or bias. The simulations allowed us to test the experimental hypotheses regarding the mechanism of DFG flip, its coupling to the ?C-helix movement, and the formation of regulatory spine. Perhaps the most significant finding is how key titratable residues, such as DFG-Asp, ?C-Glu, and HRD-Asp, change protonation states dependent on the DFG, ?C, and activation loop conformations. Our data offer direct evidence to support a long-standing hypothesis that protonation of Asp favors the DFG-out state and explain why DFG flip is also possible in simulations with deprotonated Asp. The simulations also revealed intermediate states, among which a unique DFG-out/?-C state formed as DFG-Asp is moved into a back pocket forming a salt bridge with catalytic Lys, which can be tested in selective inhibitor design. Our finding of how proton coupling enables the remarkable conformational plasticity may shift the paradigm of computational studies of kinases which assume fixed protonation states. Understanding proton-coupled conformational dynamics may hold a key to further innovation in kinase drug discovery.

SUBMITTER: Tsai CC

PROVIDER: S-EPMC6933753 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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How Electrostatic Coupling Enables Conformational Plasticity in a Tyrosine Kinase.

Tsai Cheng-Chieh CC Yue Zhi Z Shen Jana J

Journal of the American Chemical Society 20190913 38

Protein kinases are important cellular signaling molecules involved in cancer and a multitude of other diseases. It is well-known that inactive kinases display a remarkable conformational plasticity; however, the molecular mechanisms remain poorly understood. Conformational heterogeneity presents an opportunity but also a challenge in kinase drug discovery. The ability to predictively model various conformational states could accelerate selective inhibitor design. Here we performed a proton-coup ...[more]

PMID: 31476863

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Project description:Protein kinases are enzymes that catalyze the covalent transfer of the γ-phosphate of an adenosine triphosphate (ATP) molecule onto a tyrosine, serine, threonine, or histidine residue in the substrate and thus send a chemical signal to networks of downstream proteins. They are important cellular signaling enzymes that regulate cell growth, proliferation, metabolism, differentiation, and migration. Unregulated protein kinase activity is often associated with a wide range of diseases, therefore making protein kinases major therapeutic targets. A prototypical system of central interest to understand the regulation of kinase activity is provided by tyrosine kinase c-Src, which belongs to the family of Src-related non-receptor tyrosine kinases (SFKs). Although the broad picture of autoinhibition via the regulatory domains and via the phosphorylation of the C-terminal tail is well characterized from a structural point of view, a detailed mechanistic understanding at the atomic-level is lacking. Advanced computational methods based on all-atom molecular dynamics (MD) simulations are employed to advance our understanding of tyrosine kinase activation. The computational studies suggest that the isolated kinase domain (KD) is energetically most favorable in the inactive conformation when the activation loop (A-loop) of the KD is not phosphorylated. The KD makes transient visits to a catalytically competent active-like conformation. The process of bimolecular trans-autophosphorylation of the A-loop eventually locks the KD in the active state. Activating point mutations may act by slightly increasing the population of the active-like conformation, enhancing the availability of the A-loop to be phosphorylated. The Src-homology 2 (SH2) and Src-homology 3 (SH3) regulatory domains, depending upon their configuration, either promote the inactive or the active state of the kinase domain. In addition to the roles played by the SH3, SH2, and KD, the Src-homology 4-Unique domain (SH4-U) region also serves as a key moderator of substrate specificity and kinase function. Thus, a fundamental understanding of the conformational propensity of the SH4-U region and how this affects the association to the membrane surface are likely to lead to the discovery of new intermediate states and alternate strategies for inhibition of kinase activity for drug discovery. The existence of a multitude of KD conformations poses a great challenge aimed at the design of specific inhibitors. One promising computational strategy to explore the conformational flexibility of the KD is to construct Markov state models from aggregated MD data.

Project description:The calcium calmodulin-dependent protein kinase II (CaMKII) is a dodecameric holoenzyme important for encoding memory. Its activation, triggered by binding of calcium-calmodulin, persists autonomously after calmodulin dissociation. One (receiver) kinase captures and subsequently phosphorylates the regulatory domain peptide of a donor kinase forming a chained dimer as the first stage of autonomous activation. Protein dynamics simulations examined the conformational changes triggered by dimer formation and phosphorylation, aimed to provide a molecular rationale for human mutations that result in learning disabilities. Ensembles generated from X-ray crystal structures were characterized by network centrality and community analysis. Mutual information related collective motions to local fragment dynamics encoded with a structural alphabet. Implicit solvent tCONCOORD conformational ensembles revealed the dynamic architecture of inactive kinase domains was co-opted in the activated dimer but the network hub shifted from the nucleotide binding cleft to the captured peptide. Explicit solvent molecular dynamics (MD) showed nucleotide and substrate binding determinants formed coupled nodes in long-range signal relays between regulatory peptides in the dimer. Strain in the extended captured peptide was balanced by reduced flexibility of the receiver kinase C-lobe core. The relays were organized around a hydrophobic patch between the captured peptide and a key binding helix. The human mutations aligned along the relays. Thus, these mutations could disrupt the allosteric network alternatively, or in addition, to altered binding affinities. Non-binding protein sectors distant from the binding sites mediated the allosteric signalling; providing possible targets for inhibitor design. Phosphorylation of the peptide modulated the dielectric of its binding pocket to strengthen the patch, non-binding sectors, domain interface and temporal correlations between parallel relays. These results provide the molecular details underlying the reported positive kinase cooperativity to enrich the discussion on how autonomous activation by phosphorylation leads to long-term behavioural effects.

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How Electrostatic Coupling Enables Conformational Plasticity in a Tyrosine Kinase.

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How Electrostatic Coupling Enables Conformational Plasticity in a Tyrosine Kinase.

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