Dynamics of protein kinases: insights from nuclear magnetic resonance

Abstract

Protein kinases are ubiquitous enzymes with critical roles in cellular processes and pathology. As a result, researchers have studied their activity and regulatory mechanisms extensively. Thousands of X-ray structures give snapshots of the architectures of protein kinases in various states of activation and ligand binding. However, the extent of and manner by which protein motions and conformational dynamics underlie the function and regulation of these important enzymes is not well understood. Nuclear magnetic resonance (NMR) methods provide complementary information about protein conformation and dynamics in solution. However, until recently, the large size of these enzymes prevented researchers from using these methods with kinases. Developments in transverse relaxation-optimized spectroscopy (TROSY)-based techniques and more efficient isotope labeling strategies are now allowing researchers to carry out NMR studies on full-length protein kinases. In this Account, we describe recent insights into the role of dynamics in protein kinase regulation and catalysis that have been gained from NMR measurements of chemical shift changes and line broadening, residual dipolar couplings, and relaxation. These findings show strong associations between protein motion and events that control kinase activity. Dynamic and conformational changes occurring at ligand binding sites and other regulatory domains of these proteins propagate to conserved kinase core regions that mediate catalytic function. NMR measurements of slow time scale (microsecond to millisecond) motions also reveal that kinases carry out global exchange processes that synchronize multiple residues and allosteric interconversion between conformational states. Activating covalent modifications or ligand binding to form the Michaelis complex can induce these global processes. Inhibitors can also exploit the exchange properties of kinases by using conformational selection to form dynamically quenched states. These investigations have revealed that kinases are highly dynamic enzymes, whose regulation by interdomain interactions, ligand binding, and covalent modifications involve changes in motion and conformational equilibrium in a manner that can be correlated with function. Thus, NMR provides a unique window into the role of protein dynamics in kinase regulation and catalysis with important implications for drug design.

Figure 1

The architecture of protein kinases. The X-ray structure of the PKA catalytic subunit bound to ATP (black) and peptide inhibitor, PKI_5–24 (brown) (PDB 1ATP). Elements conserved among protein kinases that are needed for catalytic function are labeled, including the Gly-loop, Lys–Glu salt bridge, DFG-loop, hinge, activation loop, P + 1 loop, and catalytic base. Space filled segments indicate internal hydrophobic structural motifs, named regulatory (pink) and catalytic (yellow) “spines”.

Figure 2

Activation of Eph alters conformational dynamics in the kinase domain and juxtamembrane segment (JMS). (a) X-ray structure of the autoinhibited JMS-KD of EphB2 (PDB 1JPA), mapping residues with spectral perturbations (red/pink) accompanying activation by phosphorylation. NMR line broadening and chemical shift perturbations reflect conformational and dynamic changes from the JMS contact site propagating to the active site cleft. Sites for activating tyrosine phosphorylation (Y604, Y610 for murine EphB2) and an activating mutation (Y750) are represented with yellow sticks. (b) ¹⁵N-HSQC spectra overlaying autoinhibited JMS-KD (black) and activated EphB2 2P-JMS-KD (red), where resonances showing significant spectral perturbations are labeled. In 2P-JMS-KD, peaks corresponding to JMS residues (numbers underlined) shifted to ~8 ppm (green circle and bold arrows), revealing an order-to-disorder transition in this region upon activation. Adapted with permission from ref . Copyright 2006 EMBO Press.

Figure 3

Differential regulation of interdomain interactions by c-Abl kinase inhibitors. (a) The lowest energy structural models of the c-Abl/imatinib complex calculated from RDC and SAXS measurements are shown in different colors. Each model shows the SH3–SH2 domain oriented in an extended conformation, relative to the kinase domain. (b) The lowest energy models of the ternary c-Abl/imatinib/GNF-5 complex show a compact conformation with multiple contacts between SH3–SH2 and kinase domains. Adapted with permission from ref . Copyright 2013 National Academy of Sciences.

Figure 4

Conformational selection in binding the p38α inhibitor BIRB796. (a) X-ray structure of p38α (magenta) bound to BIRB796 (blue) (PDB 1KV2) shows selection for the inactive DFG-out conformation. (b) ¹⁵N-HSQC spectra show the Phe169 resonance, observed in p38α/BIRB796 (blue) but not observed in apo-p38α (black) or p38α/SB203580 (red) due to line broadening. Line broadening reflects altered dynamics leading to conformational exchange in the intermediate time regime. (c) X-ray structure of p38α (magenta) bound to SB203580 (red) (PDB 2EWA) shows that both DFG-in and DFG-out conformers can form. Adapted with permission from ref . Copyright 2006 John Wiley and Sons.

Figure 5

Ligand binding regulates conformational dynamics of PKA. (a) PKA backbone structure showing slow (microsecond to millisecond) dynamics of amides in the ternary PKA/AMP-PNP/peptide complex, reflected by the magnitude of relaxation caused by chemical exchange (R_ex). (b) Space filled residues (orange) localized around the active site reflect amides that synchronize to the same exchange process, modeled as conformational interconversion between open and closed states (arrow). Adapted with permission from ref . Copyright 2010 Nature Publishing Group.

Figure 6

Global motion accompanies activation of ERK2 by phosphorylation. (a) Isoleucine, leucine, and valine methyls in the kinase core of 0P-ERK2 show large variations in k_ex, reflecting uncoupled, local motions. Each methyl could be fitted individually, except those in black spheres, which show microsecond to millisecond dynamics but high errors in k_ex. (b) In contrast, methyls throughout the core of 2P-ERK2 (blue) could be fit together to a two-state exchange process with global k_ex ≈ 300 s⁻¹. Black sticks show the phosphorylation sites in the activation loop. (c) A mutation at the hinge (M106G, E107G) induces global exchange, even in the absence of phosphorylation. Adapted with permission from ref . Copyright 2014 National Academy of Sciences.