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-A 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.

Fig. 1.

Kinase expression in bacteria. (A) Sequence of the N-terminal HIS-tag and the NdeI and SalI polylinker region from pET-N6 BI-PTP, the bicistronic expression vector used for coexpression of protein tyrosine kinases with the catalytic fragment of the tyrosine phosphatase, PTP1B. (B) Kinase-encoding sequences are ligated into vectors without (pET-N6) or with (pET-N6 BI-PTP) the phosphatase-encoding sequences, for production of the phosphorylated or the unphosphorylated proteins, respectively. (C) (Top) Coomassie-stained SDS/PAGE of 1 μg of the HIS-tagged c-Abl, c-Src, or c-Met kinases after expression without (−) or with (+) phosphatase coexpression, and after purification by metal affinity chromatography. (Middle) Western blot detection of phospho-Tyr present in 10 ng of the same kinases separated by SDS/PAGE as in Top. (Bottom) Western blot detection of phospho-Tyr present in 10 μg of the unpurified soluble protein extract from the E. coli cultures used to produce the kinases in Top and Middle.

Fig. 2.

Comparison of dose-dependent inhibition of c-Abl kinase activities by Imatinib. The inhibition of the unphosphorylated (UP) c-Abl produced in E. coli using coexpression with phosphatase occurs with an IC₅₀ of 28 nM (±5 nM), compared to an IC₅₀ of 3.3 μM (±1.1 μM) for the phosphorylated c-Abl produced in E. coli without phosphatase. Points are duplicates normalized to 100% for the uninhibited kinase, with error bars representing the standard deviation of the mean.

Fig. 3.

Ribbon cartoon images of the autoinhibited c-Met kinase domain. (A) The entire kinase domain, colored by rainbow from the N terminus to the C terminus, with a white rectangle circumscribing the region of the activation loop that blocks the active site. (B) A close-up of the autoinhibitory activation loop, with a selection of the side chains shown as stick figures, depicting the more buried interactions stabilizing the inhibitory conformation. (C) A close-up with a different selection of side chains, shown as stick figures, depicting stabilizing interactions occurring in a more superficial layer.

Fig. 4.

Location of mutations identified in human cancers. The ribbon cartoon image is colored teal, with the activation loop shown in yellow. The P-loop and aC helix are indicated by arrows. Residues K1110, E1127, and D1222 are displayed as sticks. Residues found to be mutated in human cancers are displayed as spheres at their CA atoms. Mutations include V1092I (12, 14); H1094L,Y,R (14); H1106D (14); G1119V (51); M1131T (20); T1173I (17); V1188L (20); L1195V (28); V1220I (20); D1228H,N (28, 40); Y1230H,C,D (14, 19); Y1235D (16, 18, 19); K1244R (17); and M1250T,I (17, 28, 40).