Src kinase activation: A switched electrostatic network

Özkırımlı, Elif; Post, Carol Beth

doi:10.1110/ps.051999206

Cited by 71 publications

(104 citation statements)

References 70 publications

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“…The dynamics of the central Glu310-Lys295 ion pair, and the activation-loop (A-loop) region represent dynamical indicators of the kinase activation process (44,45), which we use here to address the underlying molecular mechanism for the remarkable increase in activity and loss of stability in v-Src. For the c-Src model, we find that the Glu310-Lys295 ion pair remains closed for most of the 250-ns simulation (Fig.…”

Section: Significancementioning

confidence: 99%

Conformational processing of oncogenic v-Src kinase by the molecular chaperone Hsp90

Boczek

Reefschläger

Dehling

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Hsp90 is a molecular chaperone involved in the activation of numerous client proteins, including many kinases. The most stringent kinase client is the oncogenic kinase v-Src. To elucidate how Hsp90 chaperones kinases, we reconstituted v-Src kinase chaperoning in vitro and show that its activation is ATP-dependent, with the cochaperone Cdc37 increasing the efficiency. Consistent with in vivo results, we find that Hsp90 does not influence the almost identical c-Src kinase. To explain these findings, we designed Src kinase chimeras that gradually transform c-Src into v-Src and show that their Hsp90 dependence correlates with compactness and folding cooperativity. Molecular dynamics simulations and hydrogen/deuterium exchange of Hsp90-dependent Src kinase variants further reveal increased transitions between inactive and active states and exposure of specific kinase regions. Thus, Hsp90 shifts an ensemble of conformations of v-Src toward high activity states that would otherwise be metastable and poorly populated.Cdc37 | kinase activation | metastable states | conformational ensembles | chaperone mechanism

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Section: Significancementioning

confidence: 99%

Conformational processing of oncogenic v-Src kinase by the molecular chaperone Hsp90

Boczek

Reefschläger

Dehling

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…We will now focus on the inactive conformations; therefore, we divide the DFG-in conformations of KЈ into an active and an inactive subset. In active Src, the A-loop shifts out from the catalytic site, whereas the C-helix has a more inward orientation, recruiting several side chains to the active site (6,18,(57)(58)(59). For the homologous Hck kinase, the active and inactive subsets were shown to form two distinct free energy basins (59), with a free energy difference of about ␦g ϭ 1 kcal/mol, favoring the inactive conformations (with the inactive phosphorylation pattern).…”

Section: Src/abl-binding Free Energy Difference Measures the Conformamentioning

confidence: 99%

“…In principle, this can be done by driving the protein reversibly from one conformation to the other. However, it is very difficult and expensive (36,(57)(58)(59), because many residues change positions (Fig. 1C).…”

Section: Src/abl-binding Free Energy Difference Measures the Conformamentioning

confidence: 99%

“…What is more, by combining the MD data with experimental data and thermodynamic relations, we show that the imatinib ligand actually reports on the conformational free energy surface of apo-Src. Indeed, the Src/Abl binding free energy difference ⌬⌬G bind closely approximates the free energy difference between the DFG-out and DFG-in inactive conformations of Src in the absence of imatinib (a quantity that is very hard to measure or compute directly (57)(58)(59)). This makes it difficult to engineer increased binding by chemically modifying the imatinib ligand, although it is not impossible as shown very recently (60).…”

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confidence: 99%

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Molecular Dynamics Simulations Show That Conformational Selection Governs the Binding Preferences of Imatinib for Several Tyrosine Kinases

Aleksandrov

Simonson

2010

Journal of Biological Chemistry

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Tyrosine kinases transmit cellular signals through a complex mechanism, involving their phosphorylation and switching between inactive and active conformations. The cancer drug imatinib binds tightly to several homologous kinases, including Abl, but weakly to others, including Src. Imatinib specifically targets the inactive, so-called "DFG-out" conformation of Abl, which differs from the preferred, "DFG-in" conformation of Src in the orientation of a conserved Asp-Phe-Gly (DFG) activation loop. However, recent x-ray structures showed that Src can also adopt the DFG-out conformation and uses it to bind imatinib. The Src/Abl-binding free energy difference can thus be decomposed into two contributions. Contribution i measures the different protein-imatinib interactions when either kinase is in its DFG-out conformation. Contribution ii depends on the ability of imatinib to select or induce this conformation, i.e. on the relative stabilities of the DFG-out and DFG-in conformations of each kinase. Neither contribution has been measured experimentally. We use molecular dynamics simulations to show that contribution i is very small, 0.2 ؎ 0.6 kcal/mol; imatinib interactions are very similar in the two kinases, including long range electrostatic interactions with the imatinib positive charge. Contribution ii, deduced using the experimental binding free energy difference, is much larger, 4.4 ؎ 0.9 kcal/mol. Thus, conformational selection, easy in Abl, difficult in Src, underpins imatinib specificity. Contribution ii has a simple interpretation; it closely approximates the stability difference between the DFG-out and DFG-in conformations of apo-Src. Additional calculations show that conformational selection also governs the relative binding of imatinib to the kinases c-Kit and Lck. These results should help clarify the current framework for engineering kinase signaling.

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“…In addition to evaluation of the activated and down-regulated end states, information on how the conformational change occurs and on the transition pathways connecting active and down-regulated states is required for a complete understanding of the forces that govern conformational activation. Theoretical and simulation methods can contribute by identifying probable transition pathways (Young et al 2001;Banavali and Roux 2005;Levinson et al 2006;Ozkirimli and Post 2006;Faraldo-Gómez and Roux 2007), and by providing atomic details about the underlying process and how motion of different regions is coupled. When these pathways can be probed experimentally, the combined exploration is a powerful one that can lead to new physical insights.…”

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confidence: 99%

An electrostatic network and long‐range regulation of Src kinases

et al. 2008

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The regulatory mechanism of Src tyrosine kinases includes conformational activation by a change in the catalytic domain tertiary structure and in domain-domain contacts between the catalytic domain and the SH2/SH3 regulatory domains. The kinase is activated when tyrosine phosphorylation occurs on the activation loop, but without phosphorylation of the C-terminal tail. Activation also occurs by allostery when contacts between the catalytic domain (CD) and the regulatory SH3 and SH2 domains are released as a result of exogenous protein binding. The aim of this work is to examine the proposed role of an electrostatic network in the conformational transition and to elucidate the molecular mechanism for long-range, allosteric conformational activation by using a combination of experimental enzyme kinetics and nonequilibrium molecular dynamics simulations. Salt dependence of the induction phase is observed in kinetic assays and supports the role of an electrostatic network in the transition. In addition, simulations provide evidence that allosteric activation involves a concerted motion coupling highly conserved residues, and spanning several nanometers from the catalytic site to the regulatory domain interface to communicate between the CD and the regulatory domains.

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Src kinase activation: A switched electrostatic network

Cited by 71 publications

References 70 publications

Conformational processing of oncogenic v-Src kinase by the molecular chaperone Hsp90

Conformational processing of oncogenic v-Src kinase by the molecular chaperone Hsp90

Molecular Dynamics Simulations Show That Conformational Selection Governs the Binding Preferences of Imatinib for Several Tyrosine Kinases

An electrostatic network and long‐range regulation of Src kinases

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