Transitions to catalytically inactive conformations in EGFR kinase

Abstract

The epidermal growth factor receptor (EGFR) is a key protein in cellular signaling, and its kinase domain (EGFR kinase) is an intensely pursued target of small-molecule drugs. Although both catalytically active and inactive conformations of EGFR kinase have been resolved crystallographically, experimental characterization of the transitions between these conformations remains difficult. Using unbiased, all-atom molecular dynamics simulations, we observed EGFR kinase spontaneously transition from the active to the socalled "Src-like inactive" conformation by way of two sets of intermediate conformations: One corresponds to a previously identified locally disordered state and the other to previously undescribed "extended" conformations, marked by the opening of the ATP-binding site between the two lobes of the kinase domain. We also simulated the protonation-dependent transition of EGFR kinase to another ["Asp-Phe-Gly-out" ("DFG-out")] inactive conformation and observed similar intermediate conformations. A key element observed in the simulated transitions is local unfolding, or "cracking," which supports a prediction of energy landscape theory. We used hydrogen-deuterium (H/D) exchange measurements to corroborate our simulations and found that the simulated intermediate conformations correlate better with the H/D exchange data than existing active or inactive EGFR kinase crystal structures. The intermediate conformations revealed by our simulations of the transition process differ significantly from the existing crystal structures and may provide unique possibilities for structure-based drug discovery.molten globule intermediate state | hinge unfolding | high-entropy transition state | activation loop conformations E pidermal growth factor receptor (EGFR, also known as ErbB1/Her1) kinase, the cytoplasmic kinase domain of the cell-surface receptor EGFR (1), is a widely studied protein domain and the target of a number of small-molecule drugs used to treat a variety of cancers (2). Crystal structures reveal that the active state of EGFR kinase shares characteristics common to almost all active protein kinases (3). Two inactive conformations of EGFR kinase have also been obtained from crystal structures (4, 5), one resembling the "Src-like inactive" state and one the "Asp-Phe-Gly-out" ("DFG-out") state, each of which is observed in many other protein kinases (6).Despite these structural findings, the transition pathways between the active and inactive states of EGFR kinase remain unclear. Like many other protein domains, EGFR kinase presumably adopts a multitude of intermediate conformations; identifying such conformations is crucial to a comprehensive, molecular-level understanding of the protein's dynamic behavior in solution. It is also possible that such intermediate conformations could be captured and stabilized by suitable small molecules, offering unique opportunities for drug discovery. Intermediate conformations of EGFR kinase, however, have not been directly observed experimentally. Molecular dynamics (MD)...

Dynamic architecture of a protein kinase

“…PKA belongs to the AGC family of kinases, which are typically characterized by a cis-regulatory C-terminal tail (20). Recently multiple-microsecond timescale simulations have been performed on several tyrosine kinases (21)(22)(23)(24), and a Markov model was constructed for activation of Src kinase that Significance Protein kinases represent a critically important family of regulatory enzymes. Their activity can be altered by mutations and binding events distant from the active site.…”

Phosphorylation releases constraints to domain motion in ERK2

“…Structural studies also showed an extensive hydrogen-bond network accompanied by reduced hinge flexibility in zeta-chain-associated protein kinase 70 (ZAP-70), which was proposed to maintain the kinase-inactive form (30). Finally, molecular dynamics calculations suggested local unfolding of the hinge in the catalytic domain of epidermal growth factor receptor (EGFR), which was proposed as an intermediate step toward the transition from the inactive to the active state (31,32). However, in contrast to FGFR2, ZAP-70, and EGFR, the X-ray structures of ERK2 show no significant conformational changes around the hinge upon phosphorylation (4,5).…”

Protein dynamics and function from solution state NMR spectroscopy

“…Cracking is inherently difficult to probe experimentally but there are cases when its occurrence has been inferred from simulations. Proteins suggested to undergo cracking include Adk (Whitford et al 2007(Whitford et al , 2008, epidermal growth factor receptor (Shan et al 2013), calmodulin (Tripathi & Portman, 2009) and influenza hemagglutinin . Here we will briefly summarize the computational results for Adk and cracking in relation to a reaction coordinate linking open (inactive) and closed (active) states.…”