Mutation D816V Alters the Internal Structure and Dynamics of c-KIT Receptor Cytoplasmic Region: Implications for Dimerization and Activation Mechanisms

The type III receptor tyrosine kinase (RTK) KIT plays a crucial role in the transmission of cellular signals through phosphorylation events that are associated with a switching of the protein conformation between inactive and active states. D816V KIT mutation is associated with various pathologies including mastocytosis and cancers. D816V-mutated KIT is constitutively active, and resistant to treatment with the anti-cancer drug Imatinib. To elucidate the activating molecular mechanism of this mutation, we applied a multi-approach procedure combining molecular dynamics (MD) simulations, normal modes analysis (NMA) and binding site prediction. Multiple 50-ns MD simulations of wild-type KIT and its mutant D816V were recorded using the inactive auto-inhibited structure of the protein, characteristic of type III RTKs. Computed free energy differences enabled us to quantify the impact of D816V on protein stability in the inactive state. We evidenced a local structural alteration of the activation loop (A-loop) upon mutation, and a long-range structural re-organization of the juxta-membrane region (JMR) followed by a weakening of the interaction network with the kinase domain. A thorough normal mode analysis of several MD conformations led to a plausible molecular rationale to propose that JMR is able to depart its auto-inhibitory position more easily in the mutant than in wild-type KIT and is thus able to promote kinase mutant dimerization without the need for extra-cellular ligand binding. Pocket detection at the surface of NMA-displaced conformations finally revealed that detachment of JMR from the kinase domain in the mutant was sufficient to open an access to the catalytic and substrate binding sites

Hierarchical Modeling of Activation Mechanisms in the ABL and EGFR Kinase Domains: Thermodynamic and Mechanistic Catalysts of Kinase Activation by Cancer Mutations

Structural and functional studies of the ABL and EGFR kinase domains have recently suggested a common mechanism of activation by cancer-causing mutations. However, dynamics and mechanistic aspects of kinase activation by cancer mutations that stimulate conformational transitions and thermodynamic stabilization of the constitutively active kinase form remain elusive. We present a large-scale computational investigation of activation mechanisms in the ABL and EGFR kinase domains by a panel of clinically important cancer mutants ABL-T315I, ABL-L387M, EGFR-T790M, and EGFR-L858R. We have also simulated the activating effect of the gatekeeper mutation on conformational dynamics and allosteric interactions in functional states of the ABL-SH2-SH3 regulatory complexes. A comprehensive analysis was conducted using a hierarchy of computational approaches that included homology modeling, molecular dynamics simulations, protein stability analysis, targeted molecular dynamics, and molecular docking. Collectively, the results of this study have revealed thermodynamic and mechanistic catalysts of kinase activation by major cancer-causing mutations in the ABL and EGFR kinase domains. By using multiple crystallographic states of ABL and EGFR, computer simulations have allowed one to map dynamics of conformational fluctuations and transitions in the normal (wild-type) and oncogenic kinase forms. A proposed multi-stage mechanistic model of activation involves a series of cooperative transitions between different conformational states, including assembly of the hydrophobic spine, the formation of the Src-like intermediate structure, and a cooperative breakage and formation of characteristic salt bridges, which signify transition to the active kinase form. We suggest that molecular mechanisms of activation by cancer mutations could mimic the activation process of the normal kinase, yet exploiting conserved structural catalysts to accelerate a conformational transition and the enhanced stabilization of the active kinase form. The results of this study reconcile current experimental data with insights from theoretical approaches, pointing to general mechanistic aspects of activating transitions in protein kinases

Spark Plasma Sintering as a Route for Producing In-Demand Microstructures: Application to the Tensile-Ductility Enhancement of Polycrystalline Nickel

International audienceMetallic materials exhibit properties that, besides the chemical composition, strongly depend on their microstructure; controlling their elaboration process and understanding how the microstructure will evolve, i.e. how the material will behave, are therefore of prior importance. An innovative strategy to work out texture-free large-volume bulk forms with controlled microstructures (from mono- to multimodal) for structural applications has been developed. Aside from the fundamental understanding purposes, the methodology helps level off the mechanical properties, and potentially other physical properties, by fine-tuning the microstructure. Within the bottom-up approach, it translates into the powder metallurgy route and relates more especially to spark plasma sintering as a highly suitable technique for implementing, in terms of process control, the increasingly complex structures that are being sought, in terms of material design, for optimal performances in relation with each foreseen application. In the framework of this chapter, the methodology is presented for the case of polycrystalline nickel, in relation to tensile-ductility enhancement, and refers mostly to the works performed in our research group over the past decade. At a later stage, the developed methodology is expected to open on many other innovative microstructures and corollary new physical and mechanical properties

Strengthening of Al-Fe3Al composites by the generation of harmonic structures

Abstract Strengthening of alloys can be efficiently attained by the creation of harmonic structures: bimodal microstructures generated by controlled milling of the particulate precursors, which consist of coarse-grained cores embedded in a continuous fine-grained matrix. Here, we extend the concept of harmonic structures to metal matrix composites and analyze the effectiveness of such bimodal microstructures for strengthening composites consisting of a pure Al matrix reinforced with Fe3Al particles. Preferential microstructural refinement limited to the surface of the particles, where the Fe3Al phase is progressively fragmented, occurs during ball milling of the Al-Fe3Al composite powder mixtures. The refined surface becomes the continuous fine-grained matrix that encloses macro-regions with coarser reinforcing particles in the harmonic composites synthesized during subsequent powder consolidation. The generation of the bimodal microstructure has a significant influence on the strength of the harmonic composites, which exceeds that of the conventional material by a factor of 2 while retaining considerable plastic deformation. Finally, modeling of the mechanical properties indicates that the strength of the harmonic composites can be accurately described by taking into account both the volume fraction of reinforcement and the characteristic microstructural features describing the harmonic structure