1Institute of Biochemistry and Molecular Biology, University of Wroclaw, Tamka 2, 50-137 Wroclaw, Poland and 3Department of Biochemistry, Institute for Cancer Research, The Norwegian Radium Hospital, N-0310 Oslo, Norway
4 To whom correspondence should be addressed. E-mail: otlewski{at}protein.pl
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Abstract |
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Keywords: DNA synthesis/fibroblast growth factor/MAP kinase activation/protein engineering/stability
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Introduction |
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FGF-1 acts through activation of specific cell surface fibroblast growth factor receptors (FGFRs), leading to phosphorylation of distinct intracellular proteins, activation of phospholipase C and mitogen-activated protein kinase (MAP) kinase pathway, transcription of early genes, induction of DNA synthesis and cell proliferation (Burgess and Maciag, 1989
; Johnson and Williams, 1993
; Mason, 1994
; Powers et al., 2000
). Several reports indicate that FGF-1 is able to enter the cell and that it also has an intracellular function (Imamura et al., 1994
; Wiedlocha et al., 1994
; Olsnes et al., 2003
). However, the mechanism of translocation and intracellular role of FGF-1 in many aspects remains unclear.
FGF-1 is a single polypeptide built of 154 amino acid residues after the initiator methionine is cleaved off. Full-length FGF-1 (1154) can be cleaved after lysine 15 or glycine 20 and the biological activities of the truncated forms are very similar to those of the intact protein (Burgess, 1992). The growth factor is synthesized as a cytosolic protein without a typical signal sequence and is exported out of the cell by unknown mechanism (Jackson et al., 1992
). FGF-1 exhibits a ß-trefoil structure (Blaber et al., 1996
) shared by functionally diverse proteins such as Kunitz soybean trypsin inhibitor (Sweet et al., 1974
), interleukin-1ß and -1
(Priestle et al., 1989
), plant and bacterial toxins (Tahirov et al., 1995
; Lacy et al., 1998
), amylase (Vallee et al., 1998
), xylanase (Kaneko et al., 1999
) and hisactophilin (Habazettl et al., 1992
). This fold contains 12 ß-strands which form six two-stranded ß-hairpins (Figure 1).
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The potential pharmacological use of FGF-1 can be related mainly to its mitogenic and angiogenic properties. Among others, application of FGF-1 can be important in wound healing and in cardiovascular diseases. At physiological temperature almost 50% of the protein is unfolded. It is well known that in the unfolded state proteins are much more sensitive to protease action (Hubbard, 1998; Buczek et al., 2002
). Low stability together with sensitivity to protease action question medical applications of FGF-1. Further problems with potential FGF-1 therapy could be associated with the formulation and storage of this unstable protein. As several proteins showing a ß-trefoil fold are significantly more stable than FGF-1 (Makhatadze et al., 1994
; Krokoszynska and Otlewski, 1996
; Liu et al., 2001
), rational or semirational (consensus) design of mutations that improve its stability and half-life should be possible (Lazar et al., 2003
).
In this paper, we describe point mutants of FGF-1 with increased thermodynamic stability and multiple variants, showing additive thermodynamic effects. All mutants presented preserved biological activities of the wild-type protein.
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Materials and methods |
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The consensus concept (sequence homology approach) was applied to design stabilizing mutations. This semirational approach is based on the assumption that conserved amino acids at particular positions of a homologous sequence alignment contribute more to the protein stability than residues which occur less frequently (Steipe et al., 1994). As a source of FGF-1 homologous sequences the Pfam database was chosen (Bateman et al., 2004
). This service contains an accurate and comprehensive sequence database of protein families. From each Pfam search, sequences scoring above the family specific threshold are aligned to the profile hidden Markov model automatically to make a full alignment, that contains all members in the database that can be detected. Because Pfam provides curated multiple aligned sequences, it was possible to avoid sequences with low similarity and non-homologous to FGF-1. Since this database operates on a protein set composed of SWISS-PROT and SP-TrEMBL, all homologous sequences to FGF-1 were manually checked in order to remove repeated sequences or sequence fragments and to ensure proper alignment.
Finally, FGF-1 alignment containing 140 sequences of homologous proteins from the fibroblast growth factor family was chosen. The consensus amino acid percentage occurrence derived from the alignment was calculated. Among 136 amino acids of the FGF-1 expression construct, 38 positions involved in interactions with heparin or fibroblast growth factor receptor (FGFR) were excluded from further analysis. Calculations were performed using Ligplot software (Wallace et al., 1995) and atomic coordinates of complexes of FGF-1 with heparin and FGFR 1 and 2 (Pellegrini et al., 2000
; Plotnikov et al., 2000
; Stauber et al., 2000
). Forty-four residues in FGF-1 occur with the highest frequency in analysed alignment. For the remaining sites, substitutions with the most frequent amino acids were examined using a backbone-dependent rotamer library and scoring function implemented in Swiss-PdbViewer (Guex and Peitsch, 1997
). Mutations that disrupted or altered the existing hydrogen bond network or introduced similarly charged residues at a distance closer than 6 Å were discarded. Finally, four mutants of FGF-1 which were found to fulfil the design criteria were selected: three located close to the protein surface (H21Y, H102Y, F108Y) and one within the central core of the protein (V109I) (Figure 1). In addition, L44F mutant was prepared, which had been described as the most stabilizing of those studied by Brych et al. (2001)
. As our final goal is to stabilize FGF-1 greatly by multiple substitutions, the L44F mutation was included in this study to check whether it affects biological properties of FGF-1. Amino acid frequencies for mutated positions in homologous sequences are shown in Figure 2.
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The wild-type construct in pET-3c plasmid comprised a synthetic gene for the truncated form of FGF-1 (Met-Ala-FGF-121154) was a kind gift from Professor S.Olsnes. Residues were numbered according to the system given by Burke et al. (1993): Met-Ala(6)-Asn(7)-Tyr(8)-...-Ser(138)-Ser(139)-Asp(140). The five mutations were introduced by site-directed mutagenesis using standard QuikChange protocol (Stratagene, USA) and mutagenic oligonucleotides 2730 bases in length (MWG, Germany) and confirmed by DNA sequencing. Expression of the mutants was performed at 37°C in Escherichia coli Bl21(DE3)pLysS strain (New England Biolabs, USA) using the T7 promoter system and purified on heparinSepharose CL-6B (Amersham Biosciences, USA) as described by Wiedlocha et al. (1996)
. Before thermodynamic measurements, high-salt buffer was exchanged to phosphate buffer (25 mM H3PO4, pH 7.3) using a HiTrap desalting column and ÄKTA Explorer chromatography system (Amersham Biosciences) and the protein was passed through a 0.22 µm filter. The protein concentration was determined using the molar extinction coefficient calculated as described by Pace et al. (1995)
.
Circular dichroism (CD) measurements
CD spectra were recorded in the wavelength range 195340 nm in phosphate buffer (25 mM H3PO4, pH 7.3) at 293.15 K on a Jasco J-715 spectropolarimeter. Spectra were acquired at a protein concentration of 4 x 105 M using a 10 mm (range 240340 nm) or 0.2 mm cuvette (range 195240 nm) with a slit width set to 2 nm and a response time of 1 s.
CD-monitored thermal denaturation was carried out in the presence of 1.5 M urea in phosphate buffer. Thermal scans were performed in a 10 mm cuvette, following the ellipticity at 227 nm using a response time of 16 s. A PFD 350S automatic Peltier accessory allowed continuous monitoring of the thermal transition at a constant rate of 0.25°/min. The data were analysed using PeakFit software (Jandel Scientific Software) assuming a two-state reversible equilibrium transition as described previously (Buczek et al., 2002).
Fluorescence measurements
Fluorescence spectra were measured in phosphate buffer and in phosphate buffer containing 0.7 M guanidinium chloride (GdmCl) at 294.15 K using an FP-750 spectrofluorimeter (Jasco) equipped with an ETC 272T Peltier accessory. All measurements were carried out at a protein concentration of 2 x 106 M in a 10 mm cuvette. An excitation wavelength of 280 nm was used and emission spectra were collected from 300 to 450 nm.
Thermal denaturation measurements were performed in phosphate buffer in the presence of 0.7 M GdmCl and monitored by measuring the changes in fluorescence emission intensity at 353 nm of the single Trp107 excited at 280 nm. Denaturation data were collected at a scan rate of 0.25°/min and fitted assuming a two-state reversible equilibrium transition, as described above.
Chemical denaturation of the mutants was performed by their incubation in various concentrations of GdmCl in phosphate buffer at 294.15 K for 24 h followed by fluorescence measurements (at 353 nm) in a 10 mm cuvette at the same temperature (excitation at 280 nm). The apparent free-energy change in the absence of GdmCl (G) was determined by fitting fluorescence intensity changes at a particular concentration of GdmCl to the equation given by Santoro and Bolen (1992)
.
Additivity of stabilizing mutations
In order to check whether single mutations contribute additively to the stabilization energy of FGF-1, six double, four triple and one quadruple mutants were constructed. The stabilities of all multiple variants were determined by CD- and fluorescence-monitored thermal denaturation. The difference in free energy change of unfolding between the single or multiple mutant and the wild-type at Tden (Gden) was calculated according to the equation
Gden =
Tden x
Sden, where
Tden is the difference in denaturation temperature between mutated and wild-type protein and
Sden is the entropy change of the wild-type of FGF-1 at Tden (Becktel and Schellman, 1987
).
Sden of the wild-type at Tden was calculated from the van't Hoff equation. The non-additivity energy |
Gi| was calculated as the difference between
Gden of multiple variant and the sum of corresponding
Gden values calculated for single mutants.
Measurements of DNA synthesis
The measurements of DNA synthesis were performed by monitoring [3H]thymidine incorporation in response to stimulation with the growth factor in NIH 3T3 cells (ATCC, USA) (Klingenberg et al., 1998). The cells were starved in DMEM (Bio Whittaker, USA) with 2.5 µg/ml of insulin and 2.5 µg/ml of transferrin for 24 h at 37°C. Then, increasing concentrations of FGF-1 in the presence of 10 U/ml of heparin were added and the incubation was continued for 24 h at 37°C. During the last 6 h, the cells were incubated with 1 mCi/ml of [3H]thymidine (Amersham Pharmacia Biotech, UK). Finally, the cells were treated with 5% trichloroacetic acid for 30 min, then lysed in 0.1 M NaOH and mixed with scintillation liquid. The level of radioactivity, indicating the incorporation of [3H]thymidine into DNA, was measured using a ß-liquid scintillation counter (Tri-Carb 2100TR, Packard).
MAP kinase activation
The ability of the FGF-1 mutants to activate MAP kinase pathway was performed essentially as described by Malecki et al. (2002). NIH 3T3 cells were starved in DMEM with 0.5% FCS for 24 h at 37°C. The medium was replaced with fresh DMEM0.5% FCS and the cells were preincubated for 2 h. Heparin (10 U/ml) was added and the cells were stimulated for 10 min with 2 ng/ml of FGF-1 mutant. After washing with phosphate-buffered saline (PBS), the cells were lysed with SDS sample buffer and sonicated. Samples were analysed by western blotting. Primary antibodies used were rabbit anti-MAP kinase (p42/p44) and mouse anti-phospho-MAP kinase (p42/p44) from Cell Signaling Technology (USA) and proteins were visualized after incubation with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoReasearch Laboratories, USA).
Competitive binding of FGF-1 mutants to FGFR
Cofluent NIH 3T3 cells were washed with ice-cold binding buffer (DMEM containing 50 mM HEPES, 0.2% gelatine and 10 U/ml of heparin, pH 7.4) and incubated with 67 ng of [125I]FGF-1 and increasing concentrations of unlabelled FGF-1 wild-type or FGF-1 mutants for 24 h at 4°C (Wiedlocha et al., 1996). Subsequently, the cells were washed twice with ice-cold binding buffer, once with ice-cold PBS and once with ice-cold 1 M NaCl in PBS. The cells were lysed in 0.1 M KOH and the solubilized radioactivity was measured using a gamma-counter (1261 Multigamma, LKB Wallac). Non-specific binding was estimated by the incubation of the cells in the presence of a 100-fold molar excess of unlabelled growth factor. The wild-type of FGF-1 was iodinated by the iodogen method (Fraker and Speck, 1978
).
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Results and discussion |
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In this study, we decided to use the semirational method (the homology consensus approach), which, in a simple way, exploits rapidly growing numbers of available protein sequences. The strategy is based on the assumption that the respective consensus amino acid, as inferred from an alignment of homologous amino acid sequences contributes more to the protein stability than the non-consensus amino acid. Using the protein alignment, potential stabilizing residues can be identified and then tested individually and in combination. Successful stable mutants design according to this scheme was originally demonstrated for subtilisin BPN' (Pantoliano et al., 1989) and subsequently for immunoglobulin domains (Ohage et al., 1997
), SH3 domain (Maxwell and Davidson, 1998
), tumor suppressor p53 DNA binding domain (Nikolova et al., 1998
), GroEL minichaperones (Wang et al., 1999
), WW domain (Jiang et al., 2001
) and fungal phytase (Lehmann et al., 2002
).
Using a homology study based on the FGF family, we selected five substitutions (H21Y, L44F, H102Y, F108Y, V109I) for thermodynamic analysis.
Expression and characterization of FGF-1 mutants
All FGF-1 variants were expressed with a yield of 2040 mg of pure protein from 1 l of culture (similar to the wild-type). The purity of all protein samples was verified by SDSPAGE. In addition, the molecular masses of the mutants were within 1.0 atomic mass unit of the expected molecular weights as found by electrospray ionization mass spectrometry. Conformation of the variants was checked by fluorescence and CD measurements. The fluorescence spectrum of FGF-1 is dominated by multiple tyrosine fluorescence at 305 nm since the single Trp at position 107 is surrounded by proximal pyrrole and imidazole groups and its fluorescence in the native protein is completely quenched (Copeland et al., 1991
). During unfolding, the emission maximum is shifted to 350 nm, characteristic of tryptophan, and can be conveniently used to monitor denaturation. There are no significant changes in the emission of tyrosines. The fluorescence spectra of all FGF-1 variants are almost identical with the wild-type and show an emission maximum at 305 nm in phosphate buffer and in phosphate buffer containing 0.7 M GdmCl (data not shown), proving that the tertiary interactions are well preserved.
The far- and near-CD spectra of FGF-1 mutants are similar to the spectra of the wild-type (data not shown), proving that the introduced mutations do not change the structure of the FGF-1 molecule. The far-UV CD spectra have a minimum at around 206 nm typical of ß-sheet-rich proteins (Mach et al., 1993; Srimathi et al., 2003
). The positive peak between 220 and 240 nm results from ß-turns, loops and aromatic residues (Srimathi et al., 2003
). When FGF-1 is unfolded, the positive CD signal at 228 nm is replaced by a negative signal owing to the disruption of secondary structure.
Thermal and chemical denaturation
The thermal denaturation was monitored by the CD signal at 228 nm in the presence of 1.5 M urea and by the fluorescence emission intensity at 353 nm in the presence of 0.7 M GdmCl. Addition of 0.7 M GdmCl successfully prevented both protein aggregation and accumulation of folding intermediates during heating, thus leading to a two-state reversible process (Blaber et al., 1999). It has been reported that urea is about a two times weaker denaturant than GdmCl (Myers et al., 1995
). Hence 1.5 M urea should provide an equivalent solvent composition to 0.7 M GdmCl. We decided to perform thermal denaturation in the presence of GdmCl (ionic denaturant) and urea (uncharged denaturant) to have more versatile measurement system. We found that the thermal denaturation of the wild-type and also several FGF-1 mutants (not described in this study) in the presence of GdmCl and urea monitored by CD and fluorescence provided virtually identical thermodynamic parameters.
Denaturation curves acquired from fluorescence and CD measurements are shown in Figure 3A and B, respectively. Tables I and II summarizes Tden and H values determined for all the recombinant proteins at pH 7.3 in phosphate buffer containing 0.7 M GdmCl or 1.5 M urea. It can be observed that all but one (V109I) mutation improve the thermostability of FGF-1. The Tden value for the most stable single substituted variant differs by 3.6°C (H21Y) in the presence of 0.7 M GdmCl and by 2.2°C (L44F) in the presence of 1.5 M urea from the Tden of the wild-type (Table I). The small differences in denaturation parameters can be explained by the distinct effect of GdmCl (ionic) and urea (uncharged) on the protein electrostatics (Monera et al., 1994
).
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In order to increase further the stability of FGF-1, six double, four triple and one quadruple mutants were constructed, expressed and purified. Combinations of point mutations, leading to these 11 multiple FGF-1 variants, always allowed one to obtain more stable proteins than the respective single mutants. The largest increases in Tden values determined from fluorescence-monitored denaturation for double, triple and quadruple mutants were 5.2, 6.7 and 7.8°C, respectively (Table II, Figure 3A and B). CD-determined Tden values were somewhat lower, up to 6°C.
Additivity of stabilizing mutations
To examine whether the effects of the five single stabilizing mutations are additive, 11 thermodynamic additivity cycles were constructed and analysed. In these cycles, the stability of multiple mutants was calculated from thermal denaturation data obtained for single mutants and compared with the experimental Gden values.
Gden was calculated according to the equation
Gden =
Tden x
Sden (wild-type) (Becktel and Schellman, 1987
) where
Sden of the wild-type was found, from the van't Hoff equation, to be 216 and 211 cal/mol·K for the CD- and fluorescence-monitored thermal denaturation, respectively. The errors in
Gden were estimated to be ±0.072 kcal/mol. Hence, the error in non-additivity energy calculated from additivity cycle was 0.216, 0.288 and 0.36 kcal/mol for the double, triple and quadruple mutants, respectively. The error calculations were based on the error in the
Sden (for the wild-type) value equal to ±0.016 kcal/K, which arose from an uncertainty of about ±0.1°C in Tden and ±5.0 kcal/mol in
Hden.
As calculated from these cycles, stabilizing effects of single mutations were additive comparing either fluorescence- or CD-determined Tden values. A single exception was found for H21Y/F108Y variant that was marginally non-additive, according to fluorescence-determined parameters and the error analysis described above. The additive effects are in agreement with a clear separation of mutated sites (for all substitutions CC
exceeds 9 Å) and absence of electrostatic substitutions and, hence, lack of any interactions between introduced mutations (Wells, 1990
; Dill, 1997
).
Structural analysis of FGF-1 mutant
To explain the thermodynamic data, we employed information from available spatial structures of fibroblast growth factors deposited in the PDB. Based on these structures, we anticipate that mutations introduced to FGF-1 are adopted without structural rearrangement.
The strongest stabilization was observed for the mutation H21Y. The His residue at position 21 is conserved only in 8% and the most common amino acids are Phe (36%) and Tyr (25%) (Figure 2). Two positively charged side chains of Arg35 and Lys113 are located in the close vicinity of His21. It could be expected that removing a positive charge in this region should stabilize the protein structure. Since His21 is located on the protein surface, we decided to introduce the less hydrophobic side chain of tyrosine instead of phenylalanine. Moreover, in a recently deposited FGF-12 structure (PDB code 1q1u), Tyr21 occupies a homologous position to His21 and adopts almost identical side-chain angles (Figure 4A). This residue forms a 1.97 Å hydrogen bond to the main-chain oxygen of Lys113, hence an analogous hydrogen bond is expected to stabilize H21Y mutant.
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Phe at position 108 in the wild-type occurs half as frequently as Tyr in the set of homologous sequences (Figure 2). Interestingly, Phe108 and its equivalent in FGF-2, Tyr115, adopt similar conformations in the respective crystal structures (Figure 4C). Since the F108Y substitution stabilized the mutant by 0.94 kcal/mol, it was assumed that the hydroxyl group of the Tyr108 residue could form a hydrogen bond with the side chain of Arg88.
There is a central cavity inside the FGF-1 molecule which is characteristic of the ß-trefoil family (Blaber et al., 1996; Priestle et al., 1989
). The mutations inside the protein core filling the regional cavity are well known to stabilize protein structure (Eriksson et al., 1992
, Lim et al., 1992
). It has been shown that the L44F mutation filled the microcavity adjacent to the side chain of Leu44 in the wild-type protein and stabilized the protein by 0.7 kcal/mol (Brych et al., 2001
). In this study, this mutant was also produced and the effect on stability was confirmed (Table I).
The central cavity of FGF-1 is defined by nine highly conserved, among the fibroblast growth factor family, residues. The lowest conservation is observed at position 109, where valine occurs in about 60%. The leucine side chain (the most frequent besides valine) at position 109 could eliminate both the central cavity and the microcavity between residues Val109 and Ile111 (Brych et al., 2001). However, owing to close van der Waals contacts with residue Leu73, V109L mutation causes conformational strain and has been reported to destabilize the protein by 0.6 kcal/mol (Brych et al., 2001
). We introduced an isoleucine residue at position 109. Although Ile is conserved only in 10% in the FGF-1 family sequences (Figure 2), in the FGF-4 structure it is adopted in the central cavity without structural rearrangement and conformational strain (Bellosta et al., 2001
). Ile179 in FGF-4 adopts
1 = 65°, in contrast to the conformation of valine (wild-type) and leucine (V109L mutant) side chains at position 109 in FGF-1, which show a
1 angle close to 151° (Brych et al., 2001
). Ile179 is pointed towards residues Leu96, Val137 and Phe157, which correspond to the Leu23, Leu65 and Phe85 in FGF-1 (Figure 4D). The mutation V109I in FGF-1 could be expected to increase the stability of FGF-1. However, we did not observe such an effect. It was found that the influence of this mutation on protein stability is neutral and less defective than V109L substitution.
Biological activities
The well-established mode of FGF-1 action consists in receptor binding, receptor dimerization and activation of the tyrosine kinase in the cytoplasmic part of the receptor (Burgess and Maciag, 1989), followed by activation of the Ras/Raf/Mek/MAP kinase cascade and DNA synthesis. In this work, the biological activities of the single and multiple mutants were tested using NIH 3T3 cells, which expressed functional FGF receptor. To check whether the FGF-1 mutants are biologically competent, several experiments, including stimulation of DNA synthesis, MAP kinase activation and binding to the FGF receptor, were performed.
The ability of the cells to incorporate [3H]thymidine during 24 h of stimulation was measured to determine the mitogenic activity of the mutants. The addition of each FGF-1 mutant to NIH 3T3 cells stimulated DNA synthesis generally at the same level as the addition of the wild-type (Figure 5).
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In summary, it was shown that the homology approach is a simple and effective method to increase the stability of FGF-1. Four point mutations and 11 multiple mutations presented in this study notably improved the thermostability of FGF-1 while preserving the wild-type biological function.
This study is a good starting point for improving the stability of FGF-1 in the context of its wide potential therapeutic applications. Moreover, our results confirm again the validity of the consensus approach for the successful design of thermodynamically stable mutants.
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Notes |
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Acknowledgments |
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Received April 9, 2004; revised September 15, 2004; accepted September 16, 2004.
Edited by Robin Leatherbarrow
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