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

Weiru Wang, Adhirai Marimuthu, James Tsai, Abhinav Kumar, Heike I. Krupka, Chao Zhang, Ben Powell, Yoshihisa Suzuki, Hoa Nguyen, Maryam Tabrizizad, Catherine Luu, and Brian L. West*

Plexxikon, Inc., 91 Bolivar Drive, Berkeley, CA 94710

Communicated by Sung-Hou Kim, University of California, Berkeley, CA, January 3, 2006 (received for review December 28, 2005) kinases are a large family of cell signaling mediators found that kinase samples produced in bacteria can be hetero- undergoing intensive research to identify inhibitors or modulators geneously autophosphorylated during expression in bacteria, but useful for medicine. As one strategy, small-molecule compounds that coexpression with different phosphatases works to produce that bind the with high affinity can be used to inhibit the kinases in an unphosphorylated form (8). In the current study, activity. X-ray crystallography is a powerful method to we describe in detail the production of the c-Abl, c-Src, and reveal the structures of the kinase active sites, and thus aid in the c-Met kinases using such a system. design of high-affinity, selective inhibitors. However, a limitation c-Met is the membrane receptor for hepatocyte growth factor still exists in the ability to produce purified kinases in amounts (HGF), and is important for liver development and regeneration sufficient for crystallography. Furthermore, kinases exist in differ- (ref. 9, and references therein). A link between c-Met and ent conformation states as part of their normal regulation, and the was made when it was first cloned as an , later found ability to prepare crystals of kinases in these various states also to be a truncated protein fused to the translocated promoter remains a limitation. In this study, the c-Abl, c-Src, and c-Met region as the result of a translocation (ref. 10, and kinases are produced in high yields in Escherichia coli by using a references therein). Further links to cancer have been docu-

bicistronic vector encoding the PTP1B tyrosine phosphatase. A mented through the identification of germline in the BIOCHEMISTRY 100-fold lower dose of the inhibitor, , was observed to c-Met gene in the majority of hereditary papillary renal carci- inhibit the unphosphorylated form of c-Abl kinase prepared by nomas (11, 12), and in gastric cancer (13). Somatic mutations in using this vector, compared to the phosphorylated form produced the c-Met gene have been identified in sporadic papillary renal without PTP1B, consistent with the known selectivity of this carcinomas (14), small cell lung cancer (15), squamous cell inhibitor for the unactivated conformation of the enzyme. Unphos- cancer of the oropharynx (16), hepatocellular carcinomas (17), phorylated c-Met kinase produced with this vector was used to and lung and lymph node metastases (18, 19). Such truncated obtain the crystal structure, at 2.15-Å resolution, of the autoinhib- and mutated forms of c-Met are found to transform cells in ited form of the kinase domain, revealing an intricate network of culture (18, 20), as well as to cause tumor formation in transgenic interactions involving c-Met residues documented previously to mice (21). When c-Met expression is expressed at high levels in cause dysregulation when mutated in several . mice, it loses its dependence on HGF stimulation (22). However, in the majority of cancers where c-Met plays a role, it is thought autoinhibition ͉ c-Abl ͉ c-Src ͉ cancer to be through a modest induction of c-Met expression levels, and it has been demonstrated that hypoxia can up-regulate the c-Met gene (23–25). Even with activating point mutations, the onco- equencing of the indicates there are Ͼ500 genic actions of c-Met typically still require increased expression different expressed in man (1). Many of S levels (26, 27), and remain dependent on HGF stimulation (28). these are already known to play important roles in biology, and Strategies to reduce c-Met activity include targeting both the all could potentially be important as targets for pharmaceutical extracellular receptor domain in addition to the intracellular intervention in medicine. Conservation in the active site residues domain (23–25, 29–31). within the protein kinase gene family makes the development of The c-Met receptor is composed of an extracellular alpha selective kinase inhibitors challenging. Structural biology offers chain and a transmembrane beta chain, products of a single gene valuable information useful in the design of new inhibitors (2), that become proteolytically cleaved but that remain associated but a limitation in its application to kinases can often be the through a disulfide bond (see ref. 32 for review). Crystal inability to produce highly purified in amounts suitable structures have been reported for the extracellular c-Met Sema for cocrystallography. Inhibitor binding sometimes can be sen- domain (33), as well as a mutated form of the intracellular sitive to the specific conformation state of a kinase (3), or to tyrosine kinase domain (34, 35). Signaling through c-Met is changes in the kinase sequence caused by mutations, such as thought to occur upon HGF binding through dimerization in the those occurring during cancer progression (4–7). These pose membrane (23), leading to activation of the autoinhibited re- further barriers to the implementation of structural approaches ceptor through transphosphorylation. Once phosphorylated, the to drug design, as there can be a need to produce the target intracellular domains intiate a cascade of signaling by binding to kinase in several different forms. several other proteins at a multifunctional docking site linked to Through efforts to create a robust system to produce protein kinases, we discovered that, contrary to common belief, it is possible to produce many kinases in bacteria, including catalytic Conflict of interest statement: W.W., A.M., J.T., A.K., H.I.K., C.Z., B.P., Y.S., H.N., M.T., C.L., domains of receptor tyrosine kinases (8). We discovered that and B.L.W. are shareholders of Plexxikon, Inc., a privately held company. good production systems can be developed by using Escherichia Abbreviation: HGF, hepatocyte growth factor. coli by a simple strategy involving testing many different N- and Data deposition: The atomic coordinates have been deposited in the , C-terminal boundaries for optimal expression (8). Such analyses www.pdb.org (PDB ID code 2G15). were previously difficult because of the expense of oligonucle- *To whom correspondence should be addressed. E-mail: [email protected]. otide PCR primers, but these now are readily manageable. We © 2006 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0600048103 PNAS ͉ March 7, 2006 ͉ vol. 103 ͉ no. 10 ͉ 3563–3568 Downloaded by guest on September 29, 2021 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 (Ϯ5 nM), compared to an IC50 of 3.3 ␮M(Ϯ1.1 ␮M) 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.

ities of particular mutations found in patients with hereditary forms of renal cancer. Also in this study an analysis is made of c-Abl and c-Src kinases produced through coexpression with phosphatase in E. coli. Phosphorylation-dependent differences are documented for bacterially expressed c-Abl in the sensitivity to the inhibitor, Imatinib, shown previously to inhibit preferen- tially the unphosphorylated form of c-Abl (3). Thus the use of the phosphatase coexpression system can facilitate the develop- ment of kinase inhibitor therapeutics that target different pro- tein conformation states.

Results The bicistronic pET-N6 BI-PTP plasmid (Fig. 1A) is a conve- nient vector for bacterial expression of unphosphorylated ty- Fig. 1. Kinase expression in bacteria. (A) Sequence of the N-terminal HIS-tag rosine kinases. We have engineered several kinases into both this and the NdeI and SalI polylinker region from pET-N6 BI-PTP, the bicistronic vector and the parent pET-N6 vector lacking the PTP (Fig. 1B). expression vector used for coexpression of protein tyrosine kinases with the Thus, c-Abl and c-Src coding sequences were engineered into catalytic fragment of the tyrosine phosphatase, PTP1B. (B) Kinase-encoding these vectors, choosing boundaries similar to ones described in sequences are ligated into vectors without (pET-N6) or with (pET-N6 BI-PTP) earlier structure determination reports. c-Met kinase domain the phosphatase-encoding sequences, for production of the phosphorylated also was engineered into these vectors, but the boundaries had or the unphosphorylated proteins, respectively. (C)(Top) Coomassie-stained SDS͞PAGE of 1 ␮g of the HIS-tagged c-Abl, c-Src, or c-Met kinases after previously been determined empirically by testing several dif- expression without (Ϫ) or with (ϩ) phosphatase coexpression, and after ferent N and C termini for optimal expression (8). Although all purification by metal affinity chromatography. (Middle) Western blot detec- three kinases have previously been produced by using baculo- tion of phospho-Tyr present in 10 ng of the same kinases separated by virus systems, all three were found to express well in E. coli, SDS͞PAGE as in Top.(Bottom) Western blot detection of phospho-Tyr present yielding amounts Ͼ1 mg per liter culture, convenient for making in 10 ␮g of the unpurified soluble protein extract from the E. coli cultures used preparations for crystallography. to produce the kinases in Top and Middle. The successful bacterial expression of these soluble kinases did not require the coexpression of a phosphatase; each showed high the C terminus of the kinase domain (36). The juxtamembrane enrichment after only the metal affinity purification (Fig. 1C residues linked to the N terminus of the kinase participate in Upper). However, the coexpression of the phosphatase allowed modulation of the signaling cascade through the recruitment of the preparation of kinases that were minimally phosphorylated, phosphatases (37) and ubiquitination complexes (38). Within the as determined by Western blotting with an anti-phosphotyrosine kinase domain itself, activation of the wild-type c-Met involves antibody. Whereas 10 ng of the metal affinity-purified kinases the required phosphorylation of two tyrosines in the activation gave a clear phosphotyrosine signal when no PTP was expressed, loop, occurring stepwise, first at Tyr 1235 and following at Tyr this signal was abolished when the same kinases were coex- 1234 (39). For activation of c-Met harboring oncogenic point pressed with PTP (Fig. 1C, middle panel). Similarly, when 10 ␮g mutations, the requirement for phosphorylation at Tyr 1234 can of unpurified soluble proteins from the extracted bacteria were become lost (40, 41). Such regulation likely relate to specific analyzed by Western blotting, all three kinases were shown to structural features of the kinase domain. have phosphorylated the bacterial proteins, but that the coex- In this study we present the crystal structure of unphosphor- pression of the PTP could mostly reverse this (Fig. 1C Lower). ylated c-Met kinase, revealing how this kinase can exist in an These experiments demonstrate that all three of these kinases autoinhibited form when not activated by phosphorylation. The are active during the culturing, but that coexpression of active structure reveals the likely mechanism for the oncogenic activ- PTP can reverse the phosphorylation.

3564 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0600048103 Wang et al. Downloaded by guest on September 29, 2021 Table 1. Statistics of crystallographic data and refinement the conformation that underlies the autoinhibited state. The Crystallization and data collection refinement statistics are shown in Table 1. All of the residues of the apo c-Met kinase domain are visible Unit cell dimensions, Å a ϭ 103.8 in the structure, including those of the P-loop and the activation Space group P213 loop. As expected, the structure shows the canonical bilobed Solvent content, % 54.7 protein kinase fold (Fig. 3A), but with an extra helix at the N Resolution range, Å 20–2.15 terminus, and conformations of the activation loop and P-loop Unique reflections (highest shell)* 19,665 (1,643) that are dramatically different from the reported c-Met structure Data redundancy (highest shell) 5.4 (5.1) (35). The kinase is in an inactive conformation, which causes the Completeness (highest shell), % 99.2 (100) residues important for catalysis to be too far apart for activity. I͞␴(I) (highest shell) 5.3 (0.7) Thus, the distance between Lys 1110 (the conserved residue that Rsym (highest shell), % 12.5 (55.9) stabilizes ATP phosphate binding; ref. 42) and Glu 1127 (the Refinement Statistics conserved residue from the ␣C helix that should stabilize the ␴ cut off None orientation of Lys 1110 after kinase activation, ref. 42) is 10.3 Å, Total non-hydrogen atoms 2546 and Asp 1222 (the conserved residue from the ‘‘DFG’’ motif Average B factor, Å2 35.7 known to function in stabilizing Mg binding during catalysis; ref. ͞ ͞ Rcryst Rfree,% 22.3 25.9 42), is pointed away from the blocked active site. The space rms deviation between Lys 1110 and Glu 1127 is occupied by the leading Bond lengths, Å 0.007 residues of the activation loop, with Phe 1223 from the DFG Bond angles, ° 1.097 motif projecting inwardly to form van der Waals contacts

Rsym ϭ͚͉Iavg Ϫ Ij͉͚͞ Ij. Rcryst ϭ͚͉ Fo Ϫ Fc ͉͚͞Fo, where Fo and Fc are observed with residues Met 1131, Phe 1134, Val 1139 and Phe 1200, and and calculated structure factors, respectively. Rfree was calculated from a with Leu 1225 also forming buried van der Waals contacts with randomly chosen 5% of reflections excluded from the refinement, and Rcryst residues Phe 1124 and Val 1155. As the activation loop main was calculated from the remaining 95% of reflections. The rms deviation chain becomes exposed to the surface, residue Arg 1227 forms values are the rms deviation from ideal geometry. a salt bridge with Glu 1127 (Fig. 3B), thus underscoring the *Highest shell resolution, 2.21–2.15 Å. inactive aspect of this kinase conformation. In most respects, these features of the leading residues of the BIOCHEMISTRY All three kinases produced in bacteria show activitiy in vitro activation loop are similar to those reported for a structure of c-Met bound to the alkaloid inhibitor, K252 (Protein Data Bank after the extraction and purification, as demonstrated for c-Abl code 1ROP; ref. 35). However, the subsequent residues of the (Fig. 2). When the phosphorylated c-Abl made without PTP was activation loop in the current structure adopt a conformation compared with the unphosphorylated c-Abl produced by PTP that differs strikingly, by forming a series of autoinhibitory coexpression, there was a marked difference in sensitivity to the interactions with the P-loop, the ␣C helix, and with itself. Thus, inhibitor Imatinib; the IC50 concentrations differed by 100-fold. Met 1229 projects into the ATP-binding pocket, sandwiched This result confirms the difference in sensitivies observed pre- between the P-loop residue, Phe 1089, and the side chain of Lys viously with dephosphorylated and phosphorylated c-Abl prep- 1110. The P-loop conformation is further stabilized through arations derived from baculovirus cultures (3). This result dem- interactions with the main chain of Tyr 1230 and the side chain onstrates that these kinases made in bacteria are useful for of Lys 1232, as the activation loop turns in the direction of the activity analyses, and are especially good starting points for ␣C helix. Two activation loop residues that are required to investigations of conformation-dependent activities or inhibitor become phosphorylated for activation, Tyr 1234 and Tyr 1235, selectivities. project in opposite directions, with Tyr 1234 pointing inward and We obtained the crystal structure of apo c-Met tyrosine kinase forming a hydrogen bond with catalytic residue, Glu 1127, and domain at 2.15-Å resolution with the wild-type, unphosphory- with Tyr 1235 pointing to solvent, with its phenyl side chain lated protein produced in bacteria by using PTP coexpression. sitting buried in a contour formed by the subsequent residues of The c-Met protein used for previous structures used material the activation loop. Before the activation loop traverses to the from baculovirus cultures in which unphosphorylated material lower lobe, yet another layer of autoinhibitory stabilizing inter- was obtained by mutating the tyrosines of the activation loop that actions are formed through salt bridges of Lys 1240 with Asp otherwise would become phosphorylated during the production 1228 and Asp 1231 (Fig. 3C). The remainder of the activation (34, 35), and therefore the mechanisms for autoinhibition of loop is involved in crystal packing contacts, which may contrib- c-Met kinase have not yet been fully revealed structurally. By ute to stabilizing the loop in the observed conformation. How- using the wild-type, unphosphorylated protein, we investigated ever, the extensive interactions between the activation loop with

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.

Wang et al. PNAS ͉ March 7, 2006 ͉ vol. 103 ͉ no. 10 ͉ 3565 Downloaded by guest on September 29, 2021 the inside of the active site strongly suggest this conformation could be adopted in solution. Discussion From the experience described here with c-Abl, c-Src, and c-Met, as well as from several other kinases we have tried, we know that phosphatase coexpression systems offer tremendous utility in the bacterial production of many kinases, as well as other proteins. However, we have found some kinase domains, such as those for ZAP70 and c-KIT, remain a challenge to overexpress in E. coli, most likely due to the presence of surface hydrophobic groups that make it difficult to maintain sufficient solubility. Variations of the system described here using other phospha- tases have also worked successfully to produce unphosphory- lated enzyme. When optimal N- and C-terminal boundaries are chosen, active c-Abl, c-Src, and c-Met kinases can be produced in E. coli even without the use of phosphatases, indicating that E. coli is not catastrophically sensitive to toxic effects of these kinase activities. This is at odds with a description using yeast (43), which might be explained trivially by differences in how the kinase boundaries were selected, what fusion tags were used, or the culture conditions selected, but may indicate that one or more essential eukaryotic yeast proteins are more highly sensi- tive to phosphorylation compared to E. coli. Several features of c-Met autoregulation can be understood from the autoinhibited structure presented here. In the quiescent state, neither of Tyr 1234 or 1235 are phosphorylated. This is certainly logical for Tyr 1234, because phosphorylation of this Fig. 4. Location of mutations identified in human cancers. The ribbon buried residue would be incompatible with the autoinhibited cartoon image is colored teal, with the activation loop shown in yellow. The conformation of the activation loop. By contrast, the phenolic P-loop and aC helix are indicated by arrows. Residues K1110, E1127, and D1222 hydroxyl of Tyr 1235 is solvent-exposed and predicted to be more are displayed as sticks. Residues found to be mutated in human cancers are available for phosphorylation. However, phosphorylation at Tyr displayed as spheres at their CA atoms. Mutations include V1092I (12, 14); 1235 should require some motion of the activation loop to allow 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 this residue enough freedom to serve as a substrate in a (16, 18, 19); K1244R (17); and M1250T,I (17, 28, 40). transphorylation reaction. Such a model is consistent with the observation that Tyr 1235 becomes phosphorylated ahead of Tyr 1234 during the activation process (39). However, phosphory- conceivably could indirectly destabilize the P-loop or activation lation at Tyr 1235 should only partially destabilize the autoin- loop from their autoinhibited conformations. From the current hibited conformation, because Tyr 1234 could still remain structure all these would be predicted to allow some of the buried; this is consistent with the finding that phosphorylation at control to remain intact, as has been observed. Certainly, Tyr 1234 is required in addition to that at Tyr 1235 for full however, there exist subtleties about the structures of mutated activation to occur (39). forms of c-Met that require additional studies to explain. Thus, The effects of some of the mutations found in cancer can also individual mutations are found to vary in the types of cancer they be rationalized from the autoinhibited structure (Fig. 4). Mu- promote (21). Furthermore, individual mutations can vary in tation of Tyr 1235 to Asp should be more destabilizing to the their affinities for small molecule inhibitors (6, 7). The latter autoinhibited conformation than even phosphorylation at this observations suggest that a subset of cancer patients treated with site, because this would bring the anionic charge into c-Met kinase inhibitors may become resistant, as observed with the hydrophobic space normally occupied by the Tyr phenyl side inhibitors of c-Abl and EGF kinase inhibitors (4, 5). However, chain. This mutation was incorporated into the protein crystal- from the current structure, it appears possible that some of the lized previously (34, 35), with the effect of causing the local known mutations might facilitate the search for new inhibitors, section of the activation loop to adopt a conformation that because they may mimic conformation states that exist during extends away from the active site. Mutation of Tyr 1230 to His the normal activation process. If such alternative states can be or Cys, found sporadically in cancers, should destabilize the targeted effectively, the mutated forms may provide a source of inhibitory interactions made by the activation loop with the material conducive for identifying novel inhibitory compounds. P-loop. Mutations of Asp 1228 to either Asn or His, found in It has been reported that the kinase inhibitor, Imatinib, hereditary renal cancer, should disrupt the salt bridge with Lys selectively inhibits the unphosphorylated form of c-Abl ex- 1240, and therefore partially destabilize the autoinhibited con- pressed in mammalian cell cultures (3). Our in vitro biochemical formation; this is consistent with the finding that such mutations, assays of bacterial c-Abl confirm these findings. This establishes when present in the germ line, predispose the individual to the utility of the phophatase coexpression system for identifying cancer that still requires decades to appear (44), an increase in new inhibitors that act selectively on the nonactivated, nonphos- c-Met expression (26), and stimulation by HGF (28, 39). phorylated forms of such kinases. In conjunction with studies of A pattern emerges within the set of mutations found in cancer mutated kinases and kinases activated in vitro, the expression of changes that cause a partial instability of the autoinhibited system described here should help in the discovery of kinase state, with no mutations found to date that would completely inhibitors for many clinical needs. disrupt the autoinhibition, such as would be expected for a mutation of Tyr 1234. From the current and previous structures, Materials and Methods some of the mutations found in cancer patients, including Met Plasmid Engineering. The vector (pET-N6) used for bacterial 1250 to Thr or Ile, can be mapped to locations where they expression of tyrosine kinases was engineered as a derivative of

3566 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0600048103 Wang et al. Downloaded by guest on September 29, 2021 pET28 (Novagen), by using synthetic oligonucleotides to replace same buffer. Eluted protein was fractionated with 20 ml SP and the pET28 HIS-tag and polylinker with a noncleavable N- Q FF columns (GE Healthcare) in 20 mM Tris (pH 7.5), 5% terminal HIS-tag placed upstream of an NdeI-SalI polylinker glycerol; protein flow through from SP FF column was diluted (Fig. 1A). A bicistronic vector (pET-N6 BI-PTP) that encodes into 20 mM CHES (pH 9.5) and reloaded over Q FF column. the first 283 catalytic residues of the human protein tyrosine Eluted protein was concentrated and fractionated with Super- phosphatase, PTP1B [National Center for Biotechnology Infor- dex200 26͞60 SEC (GE Healthcare) in 20 mM Tris (pH 8.8), 150 mation (NCBI) accession no. NM࿝002827), on a bicistronic mM NaCl, 5% glycerol, and 10 mM DTT. The c-Met protein mRNA was engineered by using PCR to amplify the sequences behaved monomerically and was concentrated to 20 mg͞ml and encoding PTP1B and inserting them with a second ribosome stored at Ϫ80°C. into the pET-N6 SalI polylinker site (Fig. 1A). The DNA fragments encoding the kinases analyzed here were c-Met Crystallization, Data Collection, and Structure Determination. obtained by PCR of cDNA made from human tissues (Invitrogen) Crystals of human c-Met kinase domain were grown by the by using synthetic oligonucleotides as primers. The final vectors sitting drop vapor diffusion method at 4°C. Protein solution at 16 ͞ ⅐ encoded Gly 1056 through Gly 1364 of c-Met (NCBI NM࿝000245), mg ml containing 20 mM Tris HCl (pH 8.5), 100 mM NaCl, and Gly 227 through Val 515 of c-Abl (NCBI NM࿝005157), or Val 86 14 mM 2-mercaptoethanol, was mixed with an equal volume of through Leu 536 of c-Src (NCBI NM࿝005417). Each PCR was reservoir solution containing 1.0 M diammonium hydrogen engineered to be flanked with NdeI and SalI restriction sites for phosphate, 0.2 M sodium chloride, 0.1 M citrate (pH 5.0), and ligation into the pET-N6 and pET-N6 BI-PTP vectors (Fig. 1B). 7.5% glycerol. Triangular crystals grew within 8 days to a size of 0.15-ϫ 0.15-ϫ 0.15 mm. Kinase Protein Expression. N-terminal HIS-tagged kinases for A complete x-ray diffraction data set of a c-Met crystal was small-scale analysis were produced by using E. coli strain collected at the Advanced Photon Source (APS) COM-CAT BL21(DE3) (RIL) (Stratagene) in 100 ml of 2YT media with beam line under cryogenic temperature. The diffraction data antibiotics, and induced with 0.5 mM IPTG overnight at 18°C. were integrated and scaled by using ELVES (45), MOSFLM, and Centrifuged culture pellets were extracted by sonication in 50 SCALA (46) (Table 1). mM Tris (pH between 7.0 and 8.0 depending on protein pI), 250 The structure determination with data up to 4.0 Å was successful by molecular replacement (MR) using AMORE (47) mM NaCl, 0.1% Triton X-100, 0.04 mM PMSF, 0.02% mono- with homology models from earlier kinase structures. The final thioglycerol, and 150 ␮g͞ml lysozyme. Extracts were clarified by

MR calculations used a model derived from the structure of the BIOCHEMISTRY centrifugation for 30 min at 17,000 rpm (41,800 ϫ g) in a SA600 kinase in its inactive form (Protein Data Bank rotor (Sorvall) to yield the soluble extract. HIS-tagged proteins ID code 1IRK). The space group was P2 3 with one molecule in present in the soluble extracts were purified with Talon resin 1 the asymmetric unit. The initial electron density map revealed an (BD Bioscience), with elution in 50 mM Tris (pH between 7.0 extra alpha-helix near the N terminus, and significant differences and 8.0), 100 mM NaCl, 10% glycerol, 0.04 mM PMSF, and in the P-loop and activation loop regions compared to the initial 0.02% monothioglycerol. model, which was rebuilt by using O (48). The rebuilt model was For detection of tyrosine phosphorylation of proteins from E. ␮ refined to completion (Table 1) by using CNX (49) and REFMAC5 coli,10 g of the soluble extract or 10 ng of the affinity-purified (50) against 2.15-Å data with least squares refinement, individ- HIS-tagged kinase was analyzed by Western blotting using ual B-factor refinement, and TLS refinement protocols. anti-phosphotyrosine mouse monoclonal antibody (PY100; Cell Signaling Technology) with horseradish peroxidase (HRP)-goat Biochemical Protein Phosphorylation Assays. Kinase activities were anti-mouse secondary antibody (Jackson ImmunoResearch) and determined in a reaction buffer of 50 mM Hepes (pH 7.2), 5 mM detection using ECL Plus (Amersham Pharmacia). MgCl2, 5 mM MnCl2, 0.02% BSA, 0.01% Nonidet P-40, 5% DMSO, and 10 ␮M ATP (all reagents from Sigma). The final c-Met Expression Scale-Up and Purification. c-Met kinase was pro- enzyme (c-Abl, c-Met, or c-Src) concentration was Ϸ1 ␮g͞ml. A duced in 30-liter Bioreactor cultures of E. coli strain BL21(DE3) biotinylated substrate peptide (Biotin-EEEEYEEEEYEEEEY- RIL (Stratagene) using Terrific Broth, with 15-h induction at EEEE, from New England Peptide) was used at a final concen- 12°C using 1 mM IPTG. Frozen cell pastes suspended with 40 ml tration of 200 ␮g͞ml. Twenty-microliter reactions at room of lysis buffer (100 mM potassium phosphate, pH 8.0͞250 mM temperature were initiated by ATP addition. After 30 min, the NaCl͞0.1% Igepal͞5% glycerol͞25 mM imidazole͞2mM reactions were stopped by adding 5 ␮l per well of stop buffer (50 PMSF) per liter of cells were lysed by using a microfluidizer mM Hepes, pH 7.2͞20 mM EDTA͞0.02% BSA͞0.01% Nonidet (Microfluidics M-110H) at 18,000 psi, and clarified by centrif- P-40). The extent of phosphorylation was measured by using ugation at 25,000 ϫ g at 4°C for 1 h. Supernatants were AlphScreen PY20 kits (PerkinElmer). For inhibition of c-Abl, fractionated by using Ni-Chelating Sepharose FF (GE Health- Imatinib (Novartis) was purchased and dissolved in water before care), washing with lysis buffer containing 50 mM imidazole, and dilution into the assay. by 10-column volumes of 50 mM Tris (pH 8.8), 150 mM NaCl, and 25 mM imidazole, and eluted with 500 mM imidazole in the We are grateful to Drs. J. Liu and H. Pan for critique of the manuscript.

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