Structural characterization of autoinhibited c-Met kinase produced by coexpression in bacteria with phosphatase

Abstract

Protein kinases are a large family of cell signaling mediators undergoing intensive research to identify inhibitors or modulators useful for medicine. As one strategy, small-molecule compounds that bind the active site with high affinity can be used to inhibit the enzyme activity. X-ray crystallography is a powerful method to reveal the structures of the kinase active sites, and thus aid in the design of high-affinity, selective inhibitors. However, a limitation still exists in the ability to produce purified kinases in amounts sufficient for crystallography. Furthermore, kinases exist in different conformation states as part of their normal regulation, and the ability to prepare crystals of kinases in these various states also remains a limitation. In this study, the c-Abl, c-Src, and c-Met kinases are produced in high yields in Escherichia coli by using a bicistronic vector encoding the PTP1B tyrosine phosphatase. A 100-fold lower dose of the inhibitor, Imatinib, was observed to inhibit the unphosphorylated form of c-Abl kinase prepared by using this vector, compared to the phosphorylated form produced without PTP1B, consistent with the known selectivity of this inhibitor for the unactivated conformation of the enzyme. Unphosphorylated c-Met kinase produced with this vector was used to obtain the crystal structure, at 2.15-Å resolution, of the autoinhibited form of the kinase domain, revealing an intricate network of interactions involving c-Met residues documented previously to cause dysregulation when mutated in several cancers.autoinhibition ͉ c-Abl ͉ c-Src ͉ cancer

A Drug Resistance Screen Using a Selective MET Inhibitor Reveals a Spectrum of Mutations That Partially Overlap with Activating Mutations Found in Cancer Patients

“…D1228N, Y1230C, Y1230H, and Y1230D have all been found in hereditary or sporadic PRCC (28,29), and Y1230C has also been reported in metastases of head and neck cancer (30,31). In a recently published crystal structure of the autoinhibited, completely dephosphorylated kinase domain of MET, both D1228 and Y1230 were found to be involved in interactions that stabilize the inactive conformation, providing an explanation why mutations of these residues are selected for to enhance kinase activity (32). Because of their activating nature, some of the NVP-BVU972 resistance mutations might thus confer a selective advantage even in the absence of inhibitor treatment.…”

MK-2461, a Novel Multitargeted Kinase Inhibitor, Preferentially Inhibits the Activated c-Met Receptor

“…The differences between the binding modes of MK-2461 to c-Met and to FGFR2 may contribute to the observation that MK-2461 exhibits more potent antiproliferative activity in FGFR2-driven cells than in c-Metdriven cells, despite in vitro kinase assays showing that MK-2461 is more potent against c-Met than against FGFR2. Structural studies of several unactivated tyrosine kinases revealed that parts of the activation loop and/or other structural elements assume configurations that border or block the ATP binding site (40)(41)(42)(43). Therefore, the unactivated conformation may have significantly lower affinity for ATP than does the active conformation (38,39).…”

The Insulin Receptor: Both a Prototypical and Atypical Receptor Tyrosine Kinase

“…The activation loops of ALK (anaplastic lymphoma kinase) and Met also contain three tyrosines in the same positions, but lack the aspartic acid preceding the second tyrosine (a key residue in stabilizing the pseudosubstrate conformation of InsR [Till et al 2001]), and their conformations in the unphosphorylated state differ (Wang et al 2006;Lee et al 2010). In the presence of cellular (millimolar) concentrations of Mg-ATP, the unphosphorylated activation loop probably adopts multiple conformations, including the pseudosubstrate conformation, as well as conformations in which Mg-ATP is bound and the active site is available for transphosphorylation of a tyrosine from the neighboring kinase domain.…”