Dynamic regulatory features of the protein tyrosine kinases

Abstract

The SRC, Abelson murine leukemia viral oncogene homolog 1, TEC and C-terminal SRC Kinase families of non-receptor tyrosine kinases (collectively the Src module kinases) mediate an array of cellular signaling processes and are therapeutic targets in many disease states. Crystal structures of Src modules kinases provide valuable insights into the regulatory mechanisms that control activation and generate a framework from which drug discovery can advance. The conformational ensembles visited by these multidomain kinases in solution are also key features of the regulatory machinery controlling catalytic activity. Measurement of dynamic motions within kinases substantially augments information derived from crystal structures. In this review, we focus on a body of work that has transformed our understanding of non-receptor tyrosine kinase regulation from a static view to one that incorporates how fluctuations in conformational ensembles and dynamic motions influence activation status. Regulatory dynamic networks are often shared across and between kinase families while specific dynamic behavior distinguishes unique regulatory mechanisms for select kinases. Moreover, intrinsically dynamic regions of kinases likely play important regulatory roles that have only been partially explored. Since there is clear precedence that kinase inhibitors can exploit specific dynamic features, continued efforts to define conformational ensembles and dynamic allostery will be key to combating drug resistance and devising alternate treatments for kinase-associated diseases.

Keywords: molecular conformations; protein dynamics; protein-tyrosine kinases; regulatory mechanisms.

Figure 1.

Src module kinases share the SH3-SH2-kinase cassette. (a) Domain organization of the SRC, TEC, ABL and CSK families. Regions flanking the Src module vary across kinase families. FRK and BRK are not included but are related to the SRC family. (b) Autoinhibited structure of the Src module. The structure shown is that of SRC (PDB: 2SRC), which unlike the TEC, ABL and CSK kinases contains the C-terminal phosphotyrosine tail (cyan). (c) Four SH3/proline-rich peptide complex structures (PDB: 1QWF, 1PRL, 1PRM, 1RLP) are superimposed onto the structure of autoinhibited SRC (PDB: 2SRC). The SH2-kinase linker of autoinhibited SRC is in magenta and the proline rich peptides are in gray clustered around the magenta linker. Gly254 and Leu255 from the SH2-kinase linker are labeled (alpha carbons are shown with spheres). (d) The hydrophobic stack discussed in the text is shown; W286 (distal face of N-lobe), L255 (SH2-kinase linker), and Y326 (distal face of N-lobe). Bound nucleotide and the gatekeeper residue are labeled. (e) Two views of the SRC and HCK hydrophobic stacks.

Figure 2.

SRC activation loop phosphorylation and ABL/TEC conformational gymnastics. (a) NMR peak intensity data from Seeliger (80) is mapped onto the autoinhibited SRC structure. Blue indicates increased motions (decreased peak intensities) upon activation loop phosphorylation where the ratio of the peak intensity (I) for the phosphorylated SRC kinase domain/peak intensity (I) for the unphosphorylated SRC kinase domain is less than 0.5. Red indicates decreased milli- to microsecond timescale motions (increased peak intensities) upon activation loop phosphorylation where the ratio of the peak intensity (I) for the phosphorylated SRC kinase domain/peak intensity (I) for the unphosphorylated SRC kinase domain is greater than 1.9. (b) Model showing the equilibrium between inactive and active ABL. (left) Inactive ABL: the structure of the ABL N-cap/C-cap-SH3-SH2-linker fragment (PDB: 6AMV) is shown in surface rendering for the SH3 and SH2 domains and the SH2-kinase linker is magenta (occupying the SH3 binding pocket) and the PXXP motif is unstructured. The kinase domain is indicated schematically and shows the absence of the Lys/Glu salt bridge and the inactive conformation of the C-helix and activation loop. (right) Active ABL: the ABL N-cap/C-cap-SH3-SH2-linker fragment (PDB: 6AMW) shows the alternative extended conformation where the internal PXXP motif displaces the SH2-kinase linker from the SH3 domain leading to active kinase. The Lys/Glu salt bridge forms following the conformational transition of the C-helix and the activation loop no longer adopts the closed autoinhibited conformation (dotted line indicates absence of electron density for this mobile loop. (c) Comparison of the intramolecular PXXP/SH3 complexes in ABL (PDB: 6AMW, top) and ITK (PDB: 1AWJ, bottom). In both the PXXP motif displaces the SH2-linker from the SH3 domain shifting the conformational preference toward the active state.

Protein kinase domain architecture. Key motifs that control the conformational transition between inactive and active kinase domain are shown. The study of protein dynamics is augmenting the view of kinase regulation derived from crystallography.