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Originally published In Press as doi:10.1074/jbc.M608182200 on November 28, 2006

J. Biol. Chem., Vol. 282, Issue 6, 4085-4093, February 9, 2007
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Protein Engineering of the Colony-stimulating Factor-1 Receptor Kinase Domain for Structural Studies*

Céline Schalk-Hihi1, Hong-Chang Ma, Geoffrey T. Struble, Shariff Bayoumy, Robyn Williams, Eric Devine, Ioanna P. Petrounia, Tara Mezzasalma, Lee Zeng, Carsten Schubert, Bruce Grasberger, Barry A. Springer, and Ingrid C. Deckman

From the Structural Biology, Johnson & Johnson Pharmaceuticals Research and Development, L.L.C., Exton, Pennsylvania 19341

Received for publication, August 25, 2006 , and in revised form, November 3, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 CONCLUSION
 REFERENCES
 
A parallel approach to designing crystallization constructs for the c-FMS kinase domain was implemented, resulting in proteins suitable for structural studies. Sequence alignment and limited proteolysis were used to identify and eliminate unstructured and surface-exposed domains. A small library of chimeras was prepared in which the kinase insert domain of FMS was replaced with the kinase insert domain of previously crystallized receptor-tyrosine kinases. Characterization of the newly generated FMS constructs by enzymology and thermoshift assays demonstrated similar activities and compound binding to the FMS full-length cytoplasmic domain. Two chimeras were evaluated for crystallization in the presence and absence of a variety of ligands resulting in crystal structures, and leading to a successful structure-based drug design project for this important inflammation target.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 CONCLUSION
 REFERENCES
 
Macrophage colony-stimulating factor (M-CSF,2 also known as CSF-1) stimulates macrophage proliferation, differentiation activation and survival through binding to the receptor-tyrosine kinase, FMS (also known as CSF-1R and c-fms) (1). Macrophage colony-stimulating factor is the exclusive ligand of FMS and activation of the receptor mediates the accumulation of macrophages and plays a key role in tissue remodeling (2). M-CSF expression is elevated in a number of chronic inflammatory diseases such as rheumatoid arthritis, glomerulone-phritis, and atherosclerosis (3-5). In addition, overexpression of M-CSF and FMS results in the development of preneoplastic changes and has been associated with several types of human cancers, e.g. breast, uterine, and ovarian (6). FMS levels are also increased on microglia surrounding amyloid-beta in Alzheimer's diseased brain, suggesting that overexpression of this receptor may be integral to Alzheimer's pathophysiology (7-9). These studies strongly suggest that small molecule inhibitors of FMS may prevent macrophage accumulation and suppress inflammatory responses in therapeutically important diseases.

FMS is a member of the receptor-tyrosine kinase family (RTK) and is comprised of an extracellular ligand-binding domain joined through a single membrane spanning helix to a cytoplasmic domain, composed of a juxtamembrane domain and a kinase domain (Fig. 1). The kinase domain is split by a large insertion of about 70 amino acids termed the kinase insert domain (KID). Deletion or mutation of this region in FMS revealed that the KID is not necessary for intrinsic activity but may be important for receptor internalization and degradation and for protein-protein interaction during signal transduction (10, 11). Binding of M-CSF to the extracellular domain of FMS induces receptor dimerization and autophosphorylation of tyrosines in the activation loop and in non-catalytic regions. Tyr807 (Tyr809 in human) constitutes a major autophosphorylation site and may be important for full activation of the receptor (12-15). Investigation of the murine FMS and the v-fms oncogene of feline sarcoma virus have identified three tyrosine phosphorylation sites in the KID, Tyr697, Tyr706, and Tyr721 (corresponding to Tyr699, Tyr708, and Tyr723 in the human) and two tyrosine phosphorylation sites in the juxtamembrane domain, Tyr579 and Tyr581 (Tyr546 and Tyr561 in human FMS) (10, 13, 16, 17). Phosphorylation of Tyr973 (Tyr969 in human) in the C terminus creates a binding site for ubiquitin-protein ligase, which leads to ubiquitination followed by degradation (18, 19).

A variety of RTK crystal structures have been reported, including those of the vascular endothelial growth factor receptor (VEGFR2) (20), insulin receptor (IR) (21, 22), fibroblast growth factor receptor 1 (FGFR) (23), FMS-like tyrosine kinase 3 (FLT3) (24) and others (25-30). Flexible domains can be a hindrance to crystallization and have often been deleted to generate a non-physiological subdomain more likely to crystallize. Deletion of the large KID of VEGFR2 and of FLT3 did not affect intrinsic activity but was crucial in obtaining quality diffracting crystals (20, 24). In a different approach, a flexible domain can be replaced with the more structurally characterized domain of a homolog protein by protein engineering, resulting in a chimera more suitable for structural studies. Replacement of the membrane anchoring domain of thromboxane A2 with the corresponding region of the structurally characterized P450 2C5 has been very successful in converting a membrane bound protein into a soluble chimera suitable for crystallization trials (31). Several human chimera hemoglobin subunits have also been engineered and made structural studies successful (32). Chimeric extracellular/cytoplasmic receptor-tyrosine kinases have been engineered to understand biological functions of kinases (33-39). Chimeric kinases designed to aid structural studies have however not been reported so far.

This article describes the strategy used to design optimal constructs for the crystallization and structure determination of the cytoplasmic domain of FMS. FMS was engineered into chimera proteins more suitable for crystallization trials. The modification included deletion of residues 922-972 and replacement of the FMS KID with the shorter KID of the previously crystallized RTKs, FGFR and TIE2. In addition to protein engineering, a thermal stability assay was conducted to monitor solution-dependent changes in protein stability and aggregation. The stabilizing conditions that were identified were incorporated into the FMS purification protocol to limit aggregation and improve yields. This work resulted in the successful crystal structure determination of FMS for use in the design and identification of FMS inhibitors. As inhibition of FMS kinase has broad clinical potential, the structure of this receptor will provide an important tool for structure-based design of FMS inhibitors as therapeutic agents.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 CONCLUSION
 REFERENCES
 
Materials—Oxindole, Indiburin-3' monoxime, and AG1433 were purchased from Calbiochem. AG213 and SU4312 were purchased from Tocris.

Cloning—All polymerase chain reactions (PCR) were performed with PfuUltra Hotstart DNA Polymerase (Stratagene). The products were separated on 8% agarose, gel-purified with GENECLEAN SPIN kits (Qbiogene, Inc) and cloned into pCRII (Invitrogen). After sequence confirmation, the inserts were subcloned into pDEST vector for baculovirus expression (Invitrogen).

The cytoplasmic domains of human FMS from amino acids 538-972 and 538-922 were cloned from human PC-3 cells by reverse transcriptase PCR. Oligos for PCR were designed with a SalI site and start Met prior to the FMS sequence at the 5'-end and a NotI site at the 3'-end for subcloning. PCR fragments were subcloned into a modified pDEST vector with a C-terminal His6 tag (Table 1). Chimera constructs were generated by overlapping PCR using the FMS 538-972 region as a template, and the middle fragment, which replaced the FMS KID, was synthesized (Sigma Genosys).


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  		TABLE 1
FMS constructs generated and purification yields for each construct

 
To generate FMS chimera proteins with a cleavable His tag at the N terminus, constructs were modified by PCR to generate a SalI site with Val/Asp reading frame at the 5'-end and a NotI site at the 3'-end after a stop codon. PCR fragments were sub-cloned into a modified pDEST vector with an N-terminal His6 tag and TEV cleavage site, followed by a SalI site (Table 1). All other constructs were generated by PCR and subcloned into the modified N-terminal His tag pDEST vector at the SalI/NotI sites resulting in proteins shown in Table 1.

Baculovirus Expression—All FMS constructs were expressed in Sf9 cells using Invitrogen Bac-to-Bac Baculovirus Expression System per the manual (Invitrogen).

Cell Culture—Sf9 cells were grown in ESF921 media (Expression Systems) at 27 °C.

Recombinant Production of FMS—Large scale expression was carried out in either 2-liter shake flasks or 25-liter WAVE bioreactors. Cells were maintained at no more than 4 x 106 cells/ml during growth and were monitored for >95% viability by Trypan Blue exclusion. For expression, cells were subcultured to a density of 1.6 x 106 cells/ml and infected at a multiplicity of infection (MOI) of 1. At the time of infection fetal bovine serum (Mediatech) was supplemented at a final concentration of 2%. Four hours post-infection the growth temperature was reduced to 24 °C. Cells were harvested 72-h post-infection at 1600 x g for 10 min. Viabilities were determined by Guava ViaCount or Trypan Blue and routinely were between 70 and 80% at time of harvest. Cell pellets were washed once in phosphate-buffered saline with protease inhibitors and stored at -80 °C.

Small Scale Purification to Evaluate Constructs—TheÁKTA Explorer System (GE Healthcare) was used for all purification processes. All operations were carried out at 4 °C. Thermal stability measurements were used to optimize the buffer components of the small scale purification protocol as described under "Discussion." Frozen cell pellets were thawed and resuspended in buffer A (50 mM KH2PO4-NaOH buffer, pH 7.5, 200 mM NaCl, 5% glycerol, 5 mM BME, 5 mM imidazole, 1x Complete EDTA-free protease inhibitor mixture tablets (Roche Applied Science). Resuspended cells were dounce-homogenized and mechanically lysed with an Emulsiflex-C5 (Avestin) at 10,000-15,000 psi. The extract was clarified by centrifugation at 40,000 x g for 1 h. The cleared lysate was filtered through a 0.45-µm vacuum filter and mixed gently with BD Talon metal affinity resin (BD Biosciences Clontech) overnight at 4 °C. The resin was packed into a XK column (GE Healthcare) and washed with 10-20 column volumes of buffer A, containing 10 mM imidazole followed by 10-15 column volumes of buffer A containing 20 mM imidazole. FMS was then eluted using a 10 column volume linear gradient from 10 mM to 200 mM imidazole in buffer A. Fractions were analyzed by SDS-PAGE. Fractions containing FMS were pooled and analyzed by size exclusion chromatography (Superdex 200 HR 10/30, GE Healthcare). Expression yields were determined by Bradford assay (40), using the protein assay kit from Bio-Rad according to the manufacturer's instruction with bovine serum albumin as a standard.

Large Scale Purification for Crystallization Trials—The large scale purification (from 25-30 liters of cell culture) was used to purify protein for crystallization trials. The initial steps of the purification (cell lysis and affinity purification) were performed as described for the small scale purification protocol. After affinity elution, fractions containing FMS were pooled and combined with 0.2 units of TEV Protease (Invitrogen) for each µg of FMS to remove the histidine tag. The reaction was dialyzed overnight against 50 mM KH2PO4-NaOH buffer pH 7.5, 200 mM NaCl, 5% glycerol, 2 mM glutathione, using a Spectra/Por1 membrane (with a 6-8,000 Da cut-off molecular size) for simultaneous tag removal and buffer exchange. Cleavage of the histidine tag was monitored by SDS-PAGE. After complete cleavage, FMS was incubated with 200 µl of BD Talon metal affinity resin for 2 h to remove TEV protease and the remaining histidine tag. At this stage, the yields were ~ 3 mg/liters of induced cell culture, and the protein was nearly 80% pure. Purified FMS was then filtered through a 0.2-µm cartridge filter, concentrated to 2 mg/ml using an Ultrafree-15 centrifugal filter unit (10-kDa molecular mass cut-off, Millipore) and further purified on a size exclusion column (Superdex 200 HR 10/30, GE Healthcare) in 50 mM HEPES pH 7.5, 200 mM NaCl, 2 mM DTT, 5% glycerol. Fractions containing FMS were pooled, and the protein concentration determined spectrophotometrically on a UV-Vis Spectrophotometer (PerkinElmer Life Sciences). Purified FMS was filtered through a 0.1-µm vacuum filter (Millipore), incubated with various FMS inhibitors and concentrated to a final concentration of 7-11 mg/ml using a Ultrafree membrane (10 kDa cut-off). At this stage the purity was >98% as determined by SDS-PAGE, and the protein was ready for crystallization trials.

Sequence Alignment—Protein alignment was performed in ClustalW with standard parameters (41).

Limited Proteolysis—0.1 mg of purified C-terminally tagged full-length FMS cytoplasmic domain (FMS.538-972.6His) was digested with chymotrypsin (0.035 units), trypsin (0.7 µg), thermolysin (0.05 units), subtilisin (0.0003 units), clostripain (0.01 units), elastase (0.018 units), proteinase K (0.0003 units), endopeptidase Lys-C (0.01 units), endopeptidase Glu-C (0.01 units), and endopeptidase Arg-C (0.01 units). Reactions were incubated at 37 °C in 50 mM HEPES pH 7.5, 100 mM ammonium bicarbonate, 1 mM DTT, 200 mM NaCl, 3% glycerol. For thermolysin, 5 mM calcium chloride was included into the reaction buffer. Time points were taken at 15, 30, and 60 min for each enzyme by adding the SDS-PAGE loading dye and heating at 95 °C for 5 min. The reactions were analyzed by SDS-PAGE. The N terminus of proteolytic fragments was determined by N-terminal sequencing. The PeptideCutter tool of the ExPaSy molecular biology server program was used to estimate the C terminus of proteolytic fragments that were determined to be intact at the N terminus.

N-terminal Sequencing and Peptide Mapping—Purified protein was run under denaturing conditions on a 4-12% Bis-Tris NuPage SDS-PAGE (Invitrogen). Protein was transferred onto a polyvinylidene fluoride membrane (Invitrogen) and stained with 0.025% Coomassie R250, 40% methanol. N-terminal sequencing was done by Edman degradation by Midwest Analytical, St Louis, Mo. Peptide mapping was performed by M-Scan, Inc.

Cationic Exchange—Charge homogeneity of purified protein was evaluated by cationic exchange on a Mono S HR 5/5 column (GE Healthcare). The column was pre-equilibrated in 50 mM HEPES pH 7.0, 2 mM DTT, 3% glycerol. 50 µl of purified FMS at 1 mg/ml was diluted 10-fold in the equilibration buffer before injection onto Mono S. Protein was eluted using a 10 column volume gradient from 0 to 1 M NaCl in equilibration buffer.

Activity and Inhibition by Well Known Kinase Inhibitors—The kinase inhibitors used for enzymatic characterization of the FMS constructs were purchased from Calbiochem (Oxindole, Indiburin-3'monoxime and AG1433) and from Tocris (AG213 and SU4312). Reagents used in the kinetic assay were purchased from PanVera.

The activity of the purified proteins was determined by fluorescence polarization (FP) kinetic assays. In this assay format, a fluorescein-labeled phosphopeptide (typically named tracer) and phosphopeptides or phosphoproteins generated during the FMS reaction compete for binding to an anti-phosphotyrosine antibody.

Phosphorylation of a Random Copolymer—A typical reaction solution contained 10 µg/ml poly (E4, Y), 10 µM ATP, 5 mM MgCl2, 1 mM DTT, 2 ng/µl full length FMS cytoplasmic domain (FMS.538-972.6His), or 0.4 ng/µl FMS chimera in 100 mM HEPES, pH 7.5, and detection reagent (1:1:3 mixture of anti-phosphotyrosine antibody, 10x protein-tyrosine kinase (PTK) green tracer, 10x FP dilution buffer, respectively). A control lacking ATP was included in every experiment. The kinase reaction was followed for 60 min. Polarization was read on the Analyst (Molecular Devices) using 485-nm and 530-nm excitation and emission filters, respectively.

FMS Autophosphorylation—The ability of the FMS chimeras to autophosphorylate was assessed as described above except that 100 µM ATP and 2 ng/µl of each chimera were used.

Inhibition of purified proteins by commercially available kinase inhibitors was measured by an autophosphorylation fluorescence polarization end point assay. 10 nM of the FMS full-length cytoplasmic domain (FMS.538-972.6His) or chimera was incubated with compound, 1 mM ATP and 5 mM MgCl2 in 100 mM HEPES, pH 7.5, 1 mM DTT, 0.01% (v/v) Tween-20, 2% Me2SO in a final volume of 10 µl. Control reactions were run in each plate. Assay buffer was substituted for the compound in positive and negative control wells. Positive control wells also received 6 mM EDTA. The plates were covered and incubated at room temperature for 45 min. The reaction was quenched with 6mM EDTA, and then 10 µl of the detection reagent described above were added in each well. The fluorescence polarization was measured after 30 min of incubation. Under these conditions, the polarization values for positive and negative controls were ~300 mP and ~150 mP, respectively, and were used to define the 100 and 0% inhibition of the FMS reaction. The IC50 values reported are the averages of three independent measurements.

Thermal Stability and Ligand Binding—ThermoFluor® was used to assess thermal stability of purified FMS and to measure ligand binding of several compounds (42). This technique monitors changes in the fluorescent intensity of dyes such as 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS). The fluorescent dyes are quenched in aqueous environments but increase in fluorescence on binding to the hydrophobic core of denatured proteins. Equilibrium binding ligands increase protein stability and the increase in protein melting temperature (Tm) is proportional to ligand concentration and affinity. Final melt conditions were 25 mM PIPES, pH 7.0, 100 mM NaCl, 0.1 mg/ml FMS, 100 µM 1,8-ANS. 4 µl of protein were dispensed into a well of a 384-well microplate and covered with 1 µl of silicon oil to prevent evaporation. Samples were heated at 1 °C/min. Assay plates were excited with a 390 ± 10 nm light. Emission was monitored at 470 ± 30 nm. Fluorescent intensity was measured every 1 °C as relative pixel intensity count for each well. Fluorescent intensity was plotted versus temperature and integrated to calculated melting temperatures.


Figure 1
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  		FIGURE 1.
Schematic view of FMS full-length cytoplasmic domain showing the transmembrane domain (TMR), the juxtamembrane domain (JX), the split kinase domain (K1 and K2), the kinase insert domain (KID), and the C-terminal domain (C-term). Tyrosine phosphorylation sites are also shown. Numbering of amino acid residues corresponding to various subdomains is based on the FMS sequence deposited in GenBankTM (accession no. AAH47521). Red arrows indicate limited proteolysis cleavage sites.

 


Figure 2
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  		FIGURE 2.
Size exclusion (Superdex 200 HR 10/30) elution profile. Profile of construct FMS.538-922.6His purified on Ni-NTA showing 60% aggregation (blue dotted line) and on BD TALONTM resin showing 30% aggregation (magenta full line).

 

   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
CONCLUSION
 REFERENCES
 
Construct Design Using Sequence Alignment and Limited Proteolysis—The initial construct containing the full-length cytoplasmic domain of FMS with the entire KID and a non-cleavable C-terminal His tag, FMS.538-972.6His, was generated. After expression and purification, protein yields were 2-3 mg/liter of cell culture (Table 1). Commercial (Hampton) and in-house crystallization screens were unsuccessful in producing crystals.

To design a construct more suitable for crystallization, surface-exposed loops and unstructured domains that could be a hindrance to crystallization were identified by sequence alignment and limited proteolysis. The sequence of the cytoplasmic domain of FMS.538-972 (FMSCD) was aligned with the PDB sequence of three previously crystallized receptor-tyrosine kinases (PDB: 1FGK [PDB] , 1IRK, 1FVR) (Fig. 3a) as described under "Experimental Procedures." These receptor-tyrosine kinases are similar to FMS in that they contain a KID splitting their kinase domain. FGFR, IR, and TIE2 have short KIDs that consist of 21, 16, and 19 amino acids, respectively (Fig. 3b). VEGFR2 and FLT3, also members of the receptor-tyrosine kinase family, have a much larger KID of around 80 amino acid residues. The large KIDs of VEGFR2 and FLT3 have been shown to be unstructured and their deletion was crucial for successful crystallization and structure determination (20, 24). Sequence alignment indicated that 60 residues out of the 70 amino acids composing the large KID of FMS do not align with the crystallography constructs of the other previously crystallized RTKs. In addition, FMS contains 32 residues preceding the conserved and structurally ordered catalytic core as well as a long C terminus (between 34 and 54 residues) following the last ordered amino acid of the other RTKs (Fig. 3a).

Limited proteolysis was also used to help in the design of a stable construct more likely to crystallize. Various proteases were used for this study, some with narrow specificity such as trypsin, endoproteinase Lys-C, Glu-C, and some with broad specificity such as subtilisin and thermolysin. Experiments were performed on FMS.538-972.6His as described under "Experimental Procedures." Most cleavage sites were observed within the large KID of FMS, indicating that the KID is most likely unstructured and surface exposed as had been predicted by sequence alignment (Fig. 1). Limited proteolysis with clostripain generated a 43-44 kDa band on SDS-PAGE, which upon N-terminal sequence analysis was found to be intact at its N terminus. Further analysis using the peptide cutter tool of the ExPaSy molecular biology server program identified residue 922 as the clostripain cleavage site. This data suggested that beyond amino acid 922, the C terminus of FMS is probably not well ordered. No proteolytic cleavage sites were found in the N-terminal segment of FMS, suggesting that the N terminus is probably not surface-exposed.


Figure 3
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  		FIGURE 3.
a, sequence alignment of human full-length FMS cytoplasmic domain (FMSCD), crystallized FMS. 538-922(FGFR) (FMSchim), and FGFR (1FGK), IR (1IRK), and TIE2 (1FVR) kinase domains used in crystal structure determination. Sequences used are 538-972 for FMSCD, 454-763 for FGFR1 (PDB 1FGK), 1004-1310 for IR (PDB 1IRK), and 808-1124 for TIE2 (PDB 1FVR). Sequence alignment was done with ClustalW and was based on sequence homology. Secondary structures are indicated in yellow for {alpha}-helices and in red for beta-sheets. KIDs are shown in cyan. FMSCD, FGFR, TIE2, and IR contain 74, 21, 19, and 16 amino acid residues in the KID, respectively. The first ordered residues at the N terminus and last ordered residues at the C terminus are marked in red for each kinase reported. The various N- and C-terminal truncations engineered are marked in blue in the FMSCD sequence. b, crystal structure of FGFR, TIE2, IR, and 6His.FMS.538-922(FGFR) with bound inhibitor. KIDs are highlighted in cyan.

 
A small library of constructs was designed to assess the effect of KID replacement, purification tag, and N- and C-terminal truncations on expression level, purity, aggregation, and crystallization. A total of 10 constructs were engineered (Table 1). Construct FMS.538-922.6His with a C-terminal truncation after residue 922 was designed to investigate the effect of C-terminal truncation on expression and purification. Three constructs with both N- and C-terminal deletion after residue 560, 565, and 570 at the N terminus and after residue 922 for the C terminus were designed to investigate the effect of both N- and C-terminal truncation on protein expression. Three chimeras were designed in which the large, FMS KID was replaced with the shorter KID of previously crystallized RTKs (FGFR, TIE2, and IRK), and were cloned into two truncated forms of the protein (N- and C-terminal truncation and C-terminal truncation only) (Table 1). The constructs were either N-terminally or C-terminally tagged. The N-terminal histidine tags were linked through a sequence that is recognized and cleaved by Tobacco Etch Virus (TEV) protease (43, 44) so that the tag could be removed after purification.


Figure 4
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  		FIGURE 4.
Cationic exchange elution profile (Mono S HR 5/5) showing charge heterogeneity for FMS.538-922.6His (blue dotted line) and charge homogeneity for 6His.FMS.538-922(FGFR) (magenta line) and 6His.FMS.538-922(TIE2) (brown line). Insert, SDS-PAGE showing high purity level for purified 6His.FMS.538-922(FGFR).

 


Figure 5
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  		FIGURE 5.
Comparison of activity of FMS chimeras 6His.FMS.538-922(FGFR) ({blacktriangleup}), 6His.FMS.538-922(TIE2) ({circ}), and FMS full-length cytoplasmic domain, FMS.538-972.6His ({square}). Control experiment without FMS ({diamondsuit}): (a) phosphorylation of the Poly (E4, Y) copolymer. b, autophosphorylation assay.

 
Expression, Purification, and Buffer Optimization—In an initial purification protocol, cells for FMS.538-972.6His and FMS.538-922.6His were lysed in HEPES buffer pH 7.5, 200 mM NaCl, 5% glycerol, 1 mM glutathione, 5 mM imidazole, containing protease inhibitors (1x Complete EDTA-free protease inhibitor mixture, Roche Applied Science). A nickel-based IMAC resin (Ni-NTA, Qiagen) was used for the first purification step and lysis buffer containing 200 mM imidazole was required to elute FMS from the Ni-NTA. When analyzed by size exclusion chromatography, the FMS constructs purified using this procedure were 60-80% aggregated and mostly unusable for protein crystallization (Fig. 2).

Aggregation is a major obstacle in protein purification and crystallization. Variables known to influence protein unfolding and aggregation and drive crystallization include pH, salt type, and concentration, preservatives and surfactants. Thermal shift assays (using Thermofluor®) were conducted to find optimal buffer (pH and salt) conditions and study the effect of additives and/or excipients on the stability of FMS. The assay detects small changes in the intrinsic melting temperature of proteins based on binding of ligands. Compounds that interact preferentially with the native form of the protein will increase the Tm, the temperature at which half of the protein is unfolded. Compounds that have deleterious effects on protein stability decrease the Tm (42). A systematic analysis of pH, buffer, salt type and concentration and common excipients were conducted to study their effect on FMS stability and identify conditions that should be utilized or avoided during protein production. The studies showed that Na/K phosphate buffer stabilized FMS by 1.5 degrees compared with HEPES buffer and imidazole and nickel destabilize the protein by 1.22 and 1.1 degrees at 100 mM and 0.1 mM, respectively. More details of FMS stability profiling are described elsewhere (56).

In a modified purification protocol, KH2PO4-NaOH instead of HEPES was used in the lysis buffer and the Ni-NTA was replaced by an immobilized Cobalt resin (BD TALONTM IMAC). Using the BD TALON resin, FMS could be eluted with a lower concentration of imidazole (100 mM). When analyzed by gel filtration, the FMS constructs purified under this new procedure showed only 30% aggregation (Fig. 2), indicating that incorporation of Thermofluor® stabilizing conditions into the purification protocol significantly improved protein quality. Mechanism by which the buffer components affect protein aggregation may be by direct binding to the native protein, by affecting the aggregation rate or may occur from a change in water activity. Further biophysical experiments would be needed to fully understand these mechanisms.


Figure 6
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  		FIGURE 6.
Thermoshift data showing melting transitions and two compound dose-response curves for full-length FMS cytoplasmic domain FMS.538-972.6His and two chimeras 6His.FMS.538-922(FGFR) and 6His.FMS.538-922(TIE2). a, thermal shift assay results for FMS.538-972.6His ({blacktriangleup}),6His.FMS.538-922(FGFR) ({square}), and 6His.FMS.538-922(TIE2) (bullet). The midpoint of the melting transition was 48.0 ± 0.19 °C, 48.8 ± 0.26 °C, and 49.6 ± 0.14 °C for construct 538-972.6His, 6His.TEV.FMS.538-922(FGFR), and 6His.FMS.538-922(TIE2), respectively. Apparent enthalpies values ({Delta}Happ) relating to transition slope at Tm for FMS.538-972.6His, 6His.FMS.538-922(FGFR), and 6His.FMS.538-922.6His(TIE2) are 104,364, 120,979, and 85,880 kcal/mol, respectively. b, concentration-response curves with Kd for construct FMS.538-972.6His (red line, Kd is 10 µM), 6His.FMS.538-922(FGFR) (purple line, Kd is 10 µM) and 6His.FMS.538-922(TIE2) (blue line, Kd is 12.5 µM) against JNJ31117164. Data were fit as in Ref. 55. c, concentration-response curves with Kd for construct FMS.538-972.6His (red line, Kd is 10 µM), 6His.FMS.538-922(FGFR) (purple line, Kd is 6.7 µM), and 6His.FMS.538-922(TIE2) (blue line, Kd is 8.3 µM) against staurosporine.

 
The newly engineered constructs were purified under the new buffer conditions as described in the small-scale purification protocol under "Experimental Procedures." All constructs with N-terminal truncation were found to have very low expression levels compared with the full-length FMS cytoplasmic domain construct (FMS.538-972.6His), making them difficult to purify to the level of purity required for crystallization (Table 1 and Fig. 3). All other constructs with intact N termini showed expression levels similar to FMS.538-972.6His, indicating that truncation of the N terminus affects expression. Three constructs with C-terminal truncation and KID replacement showed expression levels similar to full-length FMS cyto-plasmic domain FMS.538-972.6His. The 6His.FMS.538-922(IR) showed slightly lower expression levels and higher aggregation on size exclusion chromatography compared with the other constructs (data not shown) and was not pursued. FMS.538-922.6His and two chimeras 6His.FMS.538-922(FGFR), 6His. FMS.538-922(TIE2) was selected for further characterization.

Charge Heterogeneity—Because microheterogeneities can affect crystallization, proteins were further analyzed using cationic exchange and peptide mapping. On cationic exchange (Mono S HR 5/5), FMS.538-922.His6 eluted as several peaks (Fig. 4), indicating charge heterogeneity. Two of the three KID tyrosines (Tyr699 and Tyr723) were found partially phosphorylated by peptide mapping. The peptide fragment containing the third tyrosine (Tyr721) was not detected. The two tyrosines of the juxtamembrane domain (Tyr546 and Tyr561) and the tyrosine of the activation loop (Tyr809) showed no evidence of phosphorylation. Extensive screening with this construct generated no crystals suggesting that the large KID and/or the partial phosphorylation of the tyrosine residues in the KID are a hindrance to crystallization. This construct was abandoned for structural studies.

The two chimeras eluted as a single peak on cationic exchange as shown in Fig. 4. Replacement of the FMS KID with the KID of FGFR introduced two new tyrosine residues, neither of which was phosphorylated as determined by peptide mapping. Replacement of the FMS KID with the KID of TIE2, which contains no tyrosine residues, removed all potential phosphorylation sites in the KID. By replacing the FMS KID with the KID of FGFR and TIE2, two homogeneous FMS proteins more likely to crystallize were generated.

Enzyme Activity and Binding Affinity—To ensure that the modifications engineered did not adversely affect protein conformation, kinase activity or compound binding, the newly generated chimeras, 6His.FMS.538-922(FGFR) and 6His.FMS. 538-922(TIE2) were analyzed with enzyme and thermoshift assays and then compared with full-length FMS cytoplasmic domain, FMS.538-972.6His.

The carboxyl tail of receptor-tyrosine kinases are involved in either positive or negative regulation of tyrosine kinases (45-50). In FMS, removal of carboxyl tail Tyr969 by either mutation or truncation of the C terminus was found to increase biological activity, indicating that Tyr969 is involved in some negative regulation of kinase activity (51-54). In the enzyme assay, both chimeras showed a 20-fold higher activity than the full-length cytoplasmic domain FMS toward the copolymer poly (E4, Y) (Fig. 5a), corroborating published data. In the autophosphorylation assay, all three constructs showed a similar ability to be autophosphorylated (Fig. 5b). Inhibition by one in house inhibitor JNJ31117164 and a variety of commercially available kinase inhibitors were also determined for the FMS chimeras and compared with full-length FMS cytoplasmic domain (Table 2). All compounds showed similar IC50 values for the three constructs, indicating that the active site of the FMS chimeras was not affected by the engineered modifications.


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  		TABLE 2
IC50 of commercially available kinase inhibitors and JNJ31117164 for full-length FMS cytoplasmic domain (FMS.538-972.6His) and FMS chimeras (6His.FMS.538-922(FGFR) and 6His.FMS.538-922(TIE2))

 
Thermoshift assays were used to further compare the three constructs and confirm the data obtained by enzyme assay. The melting transitions in the absence of ligand were found to be similar with Tm values of 48.0 ± 0.19, 48.8 ± 0.26, and 49.6 ± 0.14 °C for FMS full-length cytoplasmic domain FMS.538-972.6His and the chimeras 6His.FMS.538-922(FGFR) and 6His.FMS.538-922(TIE2), respectively (Fig. 6a). Binding affinity (Kd) for JNJ31117164 and staurosporine were also measured by ThermoFluor® (42). The calculated Kd for JNJ31117164 was found to be 10 µM for FMS.538-972.6His and 6His.FMS.538-922(FGFR) and 12.5 µM for 6His.FMS.538-922(TIE2) (Fig. 6b). The calculated Kd for staurosporine was found to be 6.7 µM for 6His.FMS.538-922(FGFR) and 8.3 µM for 6His.FMS.538-922(TIE2), which compares favorably with the Kd of 10 µM measured for construct FMS.538-972.6His (Fig. 6c). These data are in good agreement with the results found by enzyme assay and indicate that the new constructs generated are sufficiently similar to the full-length FMS cytoplasmic domain to be used for structure-based drug design.

Crystallization and Crystal Structure—Both chimeras were purified using the large scale purification procedure described under "Experimental Procedures" and submitted for crystallization trials. Purity greater than 95%, as determined by SDS-PAGE, was achieved for both constructs (Fig. 4). A combination of high throughput and manual crystallization screens was used to crystallize both chimeras. Extensive screening with both apo proteins generated no crystal leads. Co-crystallization with 19 different "in house" inhibitors as well as ADP, AMP-PNP, and staurosporine was also attempted. Only one ligand, an in-house inhibitor, generated spontaneous crystallization for both the 6His.FMS.538-922(FGFR) and 6His.FMS.538-922(TIE2) chimeras. Crystallization was achieved by vapor diffusion in hanging drops with 7-10 mg/ml of FMS chimera in 15-28% PEG3350, 100 mM sodium acetate, pH 5.0-5.6, 200 mM Li2SO4, 5mM DTT, 0-3% glycerol. The successful determination of the FMS crystal structure is shown in Fig. 3b and outlined in details in the accompanying article by Schubert et al. (57) Soaking experiments, which replaced the original inhibitor with more potent compounds, were also achieved successfully.


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 CONCLUSION
REFERENCES
 
The protein engineering approach described in this manuscript allowed preparation of FMS protein suitable for crystallization. Crucial for the crystallization success was a combination of construct design, careful optimization of the purification process using thermal shift assay and high throughput crystallization with a large variety of ligands. This work is now the basis of a successful structure-based drug design project in search of FMS inhibitor for the treatment of conditions containing activated macrophages as an underlying cause of disease such as rheumatoid arthritis or cancer.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Structural Biology, Johnson & Johnson Pharmaceuticals Research and Development, L.L.C., 665 Stockton Dr., Exton, PA 19341. Tel.: 610-458-5264 x6574; Fax: 610-458-8249; E-mail: cschalkh{at}prdus.jnj.com.

2 The abbreviations used are: M-CSF, macrophage colony-stimulating factor; AMP-PNP, adenylyl imidodiphosphate; FGFR, fibroblast growth factor receptor; FLT3, FMS-like tyrosine kinase 3; FMS, colony-stimulating factor-1 receptor; FMSCD, full-length FMS cytoplasmic domain; IMAC, immobilized metal affinity chromatography; IR, insulin receptor; KID, kinase insert domain; Poly (E4, Y), Poly (Glu, Tyr) 4:1; RTK, receptor-tyrosine kinase; TEV, tobacco etch virus; TIE2, angiopoietin-1 receptor; Tm, protein melting temperature; VEGFR2, vascular endothelial growth factor; PIPES, 1,4-piperazinediethanesulfonic acid; DTT, dithiothreitol; 1,8-ANS, 1-anilinonaphthalene-8-sulfonic acid; Ni-NTA, nickel-nitrilotriacetic acid. Back


   ACKNOWLEDGMENTS
 
We thank Lawrence Kuo for critical reading of the manuscript.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 CONCLUSION
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C. Schubert, C. Schalk-Hihi, G. T. Struble, H.-C. Ma, I. P. Petrounia, B. Brandt, I. C. Deckman, R. J. Patch, M. R. Player, J. C. Spurlino, et al.
Crystal Structure of the Tyrosine Kinase Domain of Colony-stimulating Factor-1 Receptor (cFMS) in Complex with Two Inhibitors
J. Biol. Chem., February 9, 2007; 282(6): 4094 - 4101.
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