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.

Amanda Tse,Gennady M. Verkhivker,Freddie Salsbury

doi:10.1371/journal.pone.0166583

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.

Highlights

The human protein kinases are involved in regulation of many functional processes in signal transduction networks and represent one of the largest classes of clinically important therapeutic targets [1,2,3,4,5,6,7,8,9,10]
By simulating ensembles of allosteric communication pathways in the BRAF dimer complexes, we establish that paradox inducer and breaker inhibitors may activate specific signaling routes that correlate with the extent of paradoxical activation
The inactive kinase states are generally flexible and could exhibit dynamic inter-lobe movements coupled with positional changes of the αC-helix, the monomeric structure of the inactive BRAF-WT is stabilized in its dormant state through additional hydrophobic interactions between the αC-helix-out and a short helical segment of the activation loop (A-loop) [38,39]

Summary

Introduction

The human protein kinases are involved in regulation of many functional processes in signal transduction networks and represent one of the largest classes of clinically important therapeutic targets [1,2,3,4,5,6,7,8,9,10]. The inactive kinase states are often characterized by the DFG-out and closed A-loop conformations, while the active kinase forms feature the DFG-in and open A-loop conformations [20,21,22,23,24] These regions are involved in the formation of the regulatory spine (R-spine) and catalytic spine (C-spine) networks that are assembled and stabilized during conformational transformations to the active kinase states [23,24]. Dimerization-induced allosteric regulation involves coordinated transitions of the kinase domain from the inactive monomer structure to a dimer configuration in which the αC-helix moves to an active ‘in’ conformation that ensures a productive alignment of the hydrophobic spines and catalytic residues required for activation. Therapeutic and clinical utility of BRAF inhibitors that exert paradoxical activation can be diminished

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