Exploring Molecular Mechanisms of Paradoxical Activation in the BRAF Kinase Dimers: Atomistic Simulations of Conformational Dynamics and Modeling of Allosteric Communication Networks and Signaling Pathways

Abstract

The recent studies have revealed that most BRAF inhibitors can paradoxically induce kinase activation by promoting dimerization and enzyme transactivation. Despite rapidly growing number of structural and functional studies about the BRAF dimer complexes, the molecular basis of paradoxical activation phenomenon is poorly understood and remains largely hypothetical. In this work, we have explored the relationships between inhibitor binding, protein dynamics and allosteric signaling in the BRAF dimers using a network-centric approach. Using this theoretical framework, we have combined molecular dynamics simulations with coevolutionary analysis and modeling of the residue interaction networks to determine molecular determinants of paradoxical activation. We have investigated functional effects produced by paradox inducer inhibitors PLX4720, Dabrafenib, Vemurafenib and a paradox breaker inhibitor PLX7904. Functional dynamics and binding free energy analyses of the BRAF dimer complexes have suggested that negative cooperativity effect and dimer-promoting potential of the inhibitors could be important drivers of paradoxical activation. We have introduced a protein structure network model in which coevolutionary residue dependencies and dynamic maps of residue correlations are integrated in the construction and analysis of the residue interaction networks. The results have shown that coevolutionary residues in the BRAF structures could assemble into independent structural modules and form a global interaction network that may promote dimerization. We have also found that BRAF inhibitors could modulate centrality and communication propensities of global mediating centers in the residue interaction networks. By simulating allosteric communication pathways in the BRAF structures, we have determined that paradox inducer and breaker inhibitors may activate specific signaling routes that correlate with the extent of paradoxical activation. While paradox inducer inhibitors may facilitate a rapid and efficient communication via an optimal single pathway, the paradox breaker may induce a broader ensemble of suboptimal and less efficient communication routes. The central finding of our study is that paradox breaker PLX7904 could mimic structural, dynamic and network features of the inactive BRAF-WT monomer that may be required for evading paradoxical activation. The results of this study rationalize the existing structure-functional experiments by offering a network-centric rationale of the paradoxical activation phenomenon. We argue that BRAF inhibitors that amplify dynamic features of the inactive BRAF-WT monomer and intervene with the allosteric interaction networks may serve as effective paradox breakers in cellular environment.

The structural basis for Ras activation of PI3Kα lipid kinase

“…This binding shifts the equilibrium toward Raf's open state, permitting dimerization and activation of the kinase domain. 35,[60][61][62] A similar membrane recruitment and equilibrium shift scenario was also suggested for tumor suppressor RASSF5, consistent with experimental observations. 63,64 In PI3K the autoinhibition is relieved by the RTK's phosphorylated motif.…”

Raf-1 Cysteine-Rich Domain Increases the Affinity of K-Ras/Raf at the Membrane, Promoting MAPK Signaling

“…As a small p21 guanosine triphosphatase (GTPase), Ras plays a central role in cellular signal transduction pathways (Lacal et al, 1986;Lu et al, 2016a;Malumbres and Barbacid, 2003), including proliferation, differentiation, apoptosis, and senescence (Quinlan and Settleman, 2009;van Hattum and Waldmann, 2014), by binding effectors and regulators, such as Raf and phosphatidylinositol 3-kinase (PI3K) (Yan et al, 1998). Raf leads to cancer via major signaling cascades, primarily the Raf/MEK/ERK (MAPK) pathway.…”

“…Raf activation requires active Ras that anchors to the plasma membrane (PM). Membrane-anchored, active Ras binds Raf, promoting homodimerization of Raf's catalytic kinase domain and trans-autophosphorylation (Jambrina et al, 2016;Tse and Verkhivker, 2016). Active Raf dimer phosphorylates and activates MEK1/2, which induces ERK1/2 activation, with the phosphorylation signal cascading downstream.…”

Integrated Computational Approaches and Tools for Allosteric Drug Discovery

“…Computational analysis of residue interaction networks and community analysis have shown that local dynamic modules anchored around functional residues can serve as building blocks to connect distant functional regions and to mediate allosteric conformational transitions [94,252,[265][266][267][268]. Dynamic and coevolutionary residue correlations may also act as synchronizing forces that determine modular organization of allosteric interaction networks and enable efficient allosteric regulation [94,269,270]. These results have motivated the development of novel community-based methods for modelling ensembles of allosteric communication pathways in protein structures [94,269,270].…”

“…Dynamic and coevolutionary residue correlations may also act as synchronizing forces that determine modular organization of allosteric interaction networks and enable efficient allosteric regulation [94,269,270]. These results have motivated the development of novel community-based methods for modelling ensembles of allosteric communication pathways in protein structures [94,269,270]. Using this computational framework, it was found that efficient allosteric communications in various signalling proteins could be controlled by structurally stable functional centers that exploit dynamically coupled residues in their local communities to propagate cooperative structural changes.…”