Volume 42, Issue 1, Pages 9-22 (April 2011) Catalytic Control in the EGF Receptor and Its Connection to General Kinase Regulatory Mechanisms  Natalia Jura, Xuewu Zhang, Nicholas F. Endres, Markus A. Seeliger, Thomas Schindler, John Kuriyan  Molecular Cell  Volume 42, Issue 1, Pages 9-22 (April 2011) DOI: 10.1016/j.molcel.2011.03.004 Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 Activation of Protein Kinases (A) Crystal structure (PDB ID 1ATP) and cartoon representation of the active state of protein kinase A (PKA). The inset displays some of the critical components of the kinase active site: the activation loop, helix αC, P loop, ATP, magnesium ion; and the catalytic residues: DFG-aspartate (Asp 184), HRD-aspartate (Asp 166), catalytic lysine (Lys 72), catalytic glutamate (Glu 91), and the autophosphorylation site in the activation loop (Thr 197). (B) Cartoon representation of the assembly of the hydrophobic spines (the regulatory and the catalytic spine) in the active state of a kinase and of their disassembly in an inactive state. The insets to the right of the cartoons provide surface representation of the residues corresponding to the regulatory and catalytic spines in an inactive Abl kinase (PDB ID 1OPJ) and in the active Abl kinase (PDB ID 2G2I). The DFG-phenylanine is depicted, as are the catalytic lysine (K) and the catalytic glutamate in helix αC (E). Molecular Cell 2011 42, 9-22DOI: (10.1016/j.molcel.2011.03.004) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 CDK/Src-like Inactive Conformation (A) The activation mechanisms of the Src family of kinases and cyclin-dependent kinases (CDKs). When not bound to SH2 and SH3 domains, Src kinases adopt an active conformation. In contrast, CDKs are inactive when not bound to their regulators, the cyclins. Activation of CDKs requires cyclin binding. (B) Crystal structures of the Hck kinase (PDB ID 1QCF) in the CDK/Src-like inactive conformation, Lck kinase (PDB ID 3LCK) in the active conformation and the Hck kinase (PDB ID 2HCK) in an alternate CDK/Src-like inactive conformation. For clarity, the SH2 and SH3 domains are not shown in the inactive structures. Molecular Cell 2011 42, 9-22DOI: (10.1016/j.molcel.2011.03.004) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 Coupling of the DFG Flip to the CDK/Src-like Inactive Conformation (A) Two distinct conformations of the DFG motif found in the crystal structures of the Abl kinase domain. In the active structure (PDB ID 2F4J), DFG adopts the DFG-in conformation, in which the DFG-aspartate is positioned toward the active site. In the inactive, DFG-out conformation (PDB ID 1OPK), DFG-phenylalanine flips toward the active site. The DFG residues are indicated by single letter amino acid codes (D, F, G), as are the catalytic lysine (K), the catalytic glutamate in helix αC (E), and the activation loop tyrosine (Y). (B) The CDK/Src-like inactive conformation is proposed as an intermediate conformation adopted by kinases during DFG flipping. (C) A DFG-in to DFG-out flip observed in long-time scale molecular dynamics simulations. Starting from conformation 1 (active Abl kinase crystal structure, PDB ID 2F4J), the DFG-aspartate migrates away from the active site, whereas the DFG-phenylalanine enters from above. The accompanying displacement of helix αC is illustrated with conformations from the trajectory (figure adapted with permission from Shan et al. [2009]). (D) A series of kinase structures captures stepwise progression of the DFG flipping from the DFG-in to the DFG-out conformation. These steps are associated with significant movement of helix αC and a transient adoption of the CDK/Src-like inactive conformation (PDB ID IHCK [CDK2 kinase] and PDB ID 1R1W [c-Met kinase]). The images, which show helix αC, the catalytic lysine (K), the catalytic glutamate (E), the DFG-aspartate (D), and the DFG-phenylalanine (F), were generated from the coordinates indicated below each image and represent data analyzed by Shan et al. (2009). Molecular Cell 2011 42, 9-22DOI: (10.1016/j.molcel.2011.03.004) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 The CDK/Src-like Switch Underlies the Activation of the EGF Receptor Kinase Domain (A and B) Both CDKs and the EGF receptor (EGFR) kinase are stable in the inactive CDK/Src-like conformation due to a network of hydrophobic residues in the N-lobe (shown in a dot representation in magenta). During activation, these hydrophobic residues become exposed, creating an interface (an activator-binding patch or cyclin-binding patch) that binds allosteric activators of these kinases. In the case of the EGF receptor (A), one kinase domain (the activator kinase) becomes an activator of the other (the receiver kinase) by forming a head-to-tail asymmetric dimer. CDKs are subject to allosteric activation by cyclins (B). The hydrophobic residues in the EGF receptor and in cyclinA that correspond to the activator interface are shown on dot representation in dark gray. Molecular Cell 2011 42, 9-22DOI: (10.1016/j.molcel.2011.03.004) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 5 Allosteric Control of the EGF Receptor Dimerization (A) Schematic representation of EGF receptor domain organization. (B) Crystal structure and cartoon representation of the EGF receptor (EGFR) kinase domain in the presence of its full juxtamembrane segment (PDB ID 3GOP) depicts binding of the juxtamembrane latch of the receiver kinase to the activator kinase. Note that the asymmetric dimer shown here actually has the kinase domain in a CDK/Src-like inactive conformation due to mutation of the catalytic lysine residue. (C) Cartoon representation of overlapping interactions involving the juxtamembrane latch-binding site, in an active asymmetric EGFR kinase dimer (based on the structures of HER4 [PDB ID 2R4B] and EGFR [PBD ID 3GOP]). An inactive EGF receptor kinase dimer (PDB ID 3GT8), and an inactive EGF receptor kinase bound to an inhibitor Mig6 (PDB ID 2RFE). (D) A model for ligand-dependent activation of EGF receptor at the plasma membrane, which depicts contributions of the extracellular, transmembrane, and the juxtamembrane domains to receptor dimerization. Molecular Cell 2011 42, 9-22DOI: (10.1016/j.molcel.2011.03.004) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 6 HER3 Functions as an Allosteric Activator for Other Members of the EGF Receptor Family (A) Cartoon representation of possible dimerization scenarios in the EGF receptor family of kinases. (B) The crystal structures of the inactive EGF receptor kinase domain (PDB ID 3GT8) and the HER3 kinase domain (PDB ID 3KEX). The HER3 kinase domain is in the CDK/Src-like inactive conformation and adopts an altered conformation of helix αC. The DFG-aspartate is marked as D, and the DFG-phenylalanine is marked as F. Molecular Cell 2011 42, 9-22DOI: (10.1016/j.molcel.2011.03.004) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 7 A Recurring Mode for Activation of Kinases by Engaging the Helix αC Patch Crystal structures and cartoon representations of PKA (PDB ID 1ATP), the Ret receptor kinase domain (PDB ID 2IVT), the Aurora kinase (PDB ID 1Ol5), the AKT/PKB kinase (PDB ID 1O6K), the Rho-kinase (PDB ID 2V55), and the Fes kinase (PDB ID 3CD3) depict different modes by which these kinases engage the helix αC patch (HM-binding pocket in PKB/Akt) during activation. In all structures, the residues in the helix αC patch are shown in dot representation in magenta. Molecular Cell 2011 42, 9-22DOI: (10.1016/j.molcel.2011.03.004) Copyright © 2011 Elsevier Inc. Terms and Conditions