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4/5 and 8/9 -hairpin turns in human acidic fibroblast growth factor
Kasha Institute of Molecular Biophysics and Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306-4380, USA
Reprint requests to: Michael Blaber, Kasha Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306-4380, USA; e-mail: blaber{at}sb.fsu.edu; fax: (850) 644-7244.
(RECEIVED September 1, 2004; FINAL REVISION September 25, 2004; ACCEPTED September 27, 2004)
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Abstract |
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-turn is the most common type of nonrepetitive structure in globular proteins, comprising ~25% of all residues; however, a detailed understanding of effects of specific residues upon -turn stability and conformation is lacking. Human acidic fibroblast growth factor (FGF-1) is a member of the -trefoil superfold and contains a total of five -hairpin structures (antiparallel -sheets connected by a reverse turn). -Turns related by the characteristic threefold structural symmetry of this superfold exhibit different primary structures, and in some cases, different secondary structures. As such, they represent a useful system with which to study the role that turn sequences play in determining structure, stability, and folding of the protein. Two turns related by the threefold structural symmetry, the 4/5 and 8/9 turns, were subjected to both sequence-swapping and poly-glycine substitution mutations, and the effects upon stability, folding, and structure were investigated. In the wild-type protein these turns are of identical length, but exhibit different conformations. These conformations were observed to be retained during sequence-swapping and glycine substitution mutagenesis. The results indicate that the -turn structure at these positions is not determined by the turn sequence. Structural analysis suggests that residues flanking the turn are a primary structural determinant of the conformation within the turn.
Keywords: -hairpin; fibroblast growth factor; folding kinetics; stability; turn conformation
Abbreviations: FGF-1, human acidic fibroblast growth factor • NOE, nuclear Overhauser effect • NOESY, nuclear Overhauser spectroscopy • HSQC, heteronuclear single quantum correlation • TROSY, transverse-relaxation optimized spectroscopy • TOCSY, total correlation spectroscopy • ADA, N-(2-acetamido)iminodiacetic acid • DTT, dithiothreitol • GuHCl, guanidinium hydrochloride • RT, room temperature (25°C) • FGFR, fibroblast growth factor receptor • the single-letter amino acid code is utilized in the description of FGF-1 mutations • r.m.s., root-mean-square
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041094205.
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Introduction |
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-hairpins have been reported, including monomeric -hairpins (Stanger and Gellman 1998; de Alba et al. 1999a) and turns associated with a three-stranded antiparallel -sheet (Kortemme et al. 1998; Schenck and Gellman 1998; de Alba et al. 1999b). With these model systems, a close relationship was observed between turn stability and turn sequence. Furthermore, the effects of different amino acids, at specific locations within particular types of -turns, upon stability and folding are being elucidated (Gibbs et al. 2002; Blandl et al. 2003; Kim et al. 2003; Rotondi and Gierasch 2003). However, more than 50% of turns have at least one residue in common with related turn types (Hutchinson and Thornton 1994); thus, the larger structural context within which turn sequences exist appears to be key to understanding their effects upon structure, thermodynamics, and folding kinetics.
Human acidic fibroblast growth factor (FGF-1) has a total of 12 -strands, forming a series of five -hairpin structures characteristic of the -trefoil superfold (Fig. 1
; Murzin et al. 1992; Orengo et al. 1994). Among these five turns, 4/5 (i.e., connecting -strands 4 and 5) and 8/9 are related by the threefold tertiary structure symmetry inherent in the -trefoil architecture. Turn 4/5 has been argued to contribute to different binding specificities of FGF homologous factors (Olsen et al. 2003). This turn in FGF-1 has two overlapping conformations: a type IV turn (an approximate 180° turn in the main chain direction, but residues i+1 and i+2 within the turn do not exhibit characteristic 
, 
angles) spanning residue positions 48–51 (i.e., residues i through i+3 of the turn) and a type II turn (i+1 ~–60° ~+120°, i+2 ~+90° ~0°) spanning residue positions 50–53 (Hutchinson and Thornton 1996). However, based on the crystal structure (Blaber et al. 1996) residue positions 49–52 within turn 4/5 correspond to residues 90–93 within turn 8/9 (Fig. 2). In this regard, there are no insertions or deletions when comparing the main chain regions that comprise turns 4/5 and 8/9, yet these turns exhibit significantly different turn conformations. Residue positions 90–93 in turn 8/9 are defined as a type I turn (type I -turn plus G1-type -bulge; i+1 ~ –60° ~–30°, i+2 ~ –90° ~0°). The importance of this particular turn upon the protein stability and function has been discussed in several reports. An extensive contact between turn 8/9 and fibroblast growth factor receptor (FGFR) has been described (Pellegrini et al. 2000). Alternate conformations for the 8/9 turn have been reported, and have been proposed as a possible basis of various functionalities (Hecht et al. 2001; Fernandez-Tornero et al. 2003); however, a type I turn conformation for this region appears to be adopted in the absence of ligand interactions or crystal packing influences (Kim et al. 2002).
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4/5 and 8/9 have different primary structures within the structurally heterogeneous region (S50, V51, G52 in turn 4/5, and E91, N92, H93 in turn 8/9) (Fig. 1
), and an important question is whether the different turn conformations are a consequence of the heterogeneous primary structures within these regions. Furthermore, do the different turn conformations exert differential contributions toward the stability and folding of the protein? In an attempt to answer these questions, we constructed a series of mutations designed to exchange the primary structures of both turns. Furthermore, we also constructed a series of glycine mutations within these turn regions to evaluate the nature of strain inherent within the turns. Folding kinetics, stability studies, and X-ray crystallographic and NMR analyses were used to quantify the effects of these mutations. The results indicate that the residues within the turns are not the primary determinant of the turn structure, but do have a significant influence on protein stability and folding. The primary determinants of the turn structure reside within the local environment, and in particular, are postulated to involve residue positions that flank the turns. |
Results |
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8/9 turn primary structure into the 4/5 turn8/9 turn primary structure into the 4/5 turn, including V51N and G52H point mutants, S50E/V51N double mutant, and S50E/V51N/G52H triple mutant. The V51N point mutant and the S50E/V51N double mutant had no effect on protein stability (Table 1) or folding kinetics (Table 2). However, the G52H point mutation destabilized the protein by 5.5 kJ/mol, essentially due to a fourfold reduction in the folding rate (Table 2). The S50E/V51N/G52H triple mutant destabilized the protein by 3.6 kJ/mol, again, primarily due to a decrease (approximately twofold) in the rate of folding.
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4/5 turn primary structure into the 8/9 turn4/5 turn primary structure into the 8/9 turn, including a previously described H93G point mutant (Kim et al. 2002), an E91S/N92V double mutant, and an E91S/N92V/H93G triple mutant. The E91S/N92V double mutant destabilized the protein by 5.7 kJ/mol (Table 1). This destabilizing effect is due to both a fivefold reduction in the rate of folding and a threefold increase in the rate of unfolding (Table 2). The E91S/N92V/H93G triple mutant destabilized the protein by 2.8 kJ/mol, and is due primarily to a twofold increase in the rate of unfolding with only a slight decrease in the rate of folding (Table 2). Relative to the H93G mutant, the triple mutant E91S/N92V/H93G was destabilized by a substantial 9.6 kJ/mol, due to both an 18-fold reduction in the rate of folding and an approximately fourfold increase in the rate of unfolding (Table 2).
Glycine substitutions in the 4/5 and 8/9 turn regions
Three different combination glycine mutations (E49G, S50G/V51G, and E49G/S50G/V51G) were created within the 4/5 turn. All three mutants were marginally more stable than the wild-type protein, with 
G values ranging from –0.4 to –1.3 kJ/mol (Table 1). The unfolding rate for all three mutants was essentially unaffected, and the observed modest increases in stability are due primarily to increases in the rate of folding (Table 2). In reference to the G52H background, the introduction of glycine residues at positions 50 and 51 resulted in a slight increase in stability of –2.1 kJ/mol, due primarily to a twofold increase in the rate of folding. E91G, N92G, and H93G point mutations as well as an E91G/N92G double mutant in FGF-1 were characterized as part of a prior study of the 8/9 loop region (Kim et al. 2002), and a combination triple glycine mutation, E91G/N92G/H93G, is reported here. The introduction of three glycine substitutions in the 8/9 turn resulted in an increase in stability of –2.6 kJ/mol in comparison to the wild-type protein. As with the glycine mutations in the 4/5 turn, this increase in stability is due almost exclusively to an increase in the rate of folding, while the rate of unfolding is largely unaffected.
X-ray structure determinations
The V51N point mutation, S50G/V51G double mutant, and S50E/V51N double mutant yielded diffraction quality crystals, each in space group C2221, and produced high-resolution data sets with good completion (Table 3). Each structure refined to similar values of Rfree (21.4%–22.7%) and Rcryst (18.5%–19.7%) and with appropriate stereochemistry. The 2Fo – Fc difference electron density was unambiguous at the mutation site(s), and the mutant structures could be accurately modeled in each case.
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compares the backbone HN-HN NOE pattern obtained in a 3D 15N-separated NOESY-HSQC spectrum for the residues found in the 4/5 and 8/9 turns within the E91S/N92V/H93G triple mutant. The NOE cross-peaks between residues 52 and 53, combined with the lack of cross-peaks between residues 51 and 52, are consistent with a type II turn for residue positions 50–53 (as identified from the X-ray structure [Blaber et al. 1996]). Despite the sequence identity within the 8/9 turn region for the E91S/N92V/H93G triple mutant, the NOE cross-peaks between residues 91 and 92 and between 92 and 93 confirm a type I turn for residue positions 90–93 (identical to the wild-type 8/9 turn).
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Discussion |
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4/5 and 8/9 turn regions (i.e., positions 50–52 and 91–93, respectively) differ in conformation, the adjacent -sheet residues (43–49, 53–59 and 84–90, 94–100) exhibit a high degree of structural identity, and the main chain atoms overlay with an r.m.s. deviation of 0.6 Å (Fig. 2). The substitution of the 4/5 turn primary structure into the 8/9 turn, and the converse substitution of the 8/9 turn primary structure into the 4/5 turn, were observed to destabilize the protein in both cases. The degree of destabilization was of similar magnitude (2.8 and 3.6 kJ/mol, respectively), although it was achieved by differential effects on folding and unfolding rates (i.e., the mutation of the 4/5 turn destabilized primarily through a decrease in the folding rate, whereas the mutation of the 8/9 turn destabilized primarily through an increase in the unfolding rate). These results indicate that the primary structure within these turns, in both cases, is optimized to interact with the local structural environment.
Given that the 4/5 and 8/9 turn regions exhibit different conformations in the wild-type protein, an important question is whether the swapping of primary structure within these turns results in a corresponding conformational change. A crystal structure was obtained for the S50E/V51N double mutant that converts the i+1 and i+2 residues within the 4/5 turn to the equivalent 8/9 primary structure. These mutations resulted in no significant structural change to the 4/5 turn (Fig. 4). The H93G point mutation structure was reported previously (Kim et al. 2002), and results in no conformational change to the 8/9 turn. Thus, the same sequence E(i+1), N(i+2), and G(i+3) introduced into the 4/5 turn adopts the characteristic overlapping type IV and type II turns, but when introduced into the 8/9 turn adopts the characteristic type I turn. NMR spectra were used to further analyze the conformation of the 8/9 turn with the E91S/N92V/H93G triple mutant (i.e., introducing the 4/5 primary structure into the 8/9 turn). An observed intense NOE between residue 91 (i+1) and 92 (i+2) eliminates the possibility that the 8/9 turn adopts a type II or type II' conformation. Likewise, an observed intense NOE between 92 (i+2) and 93 (i+3) eliminates the possibility that a type I' is formed in the 8/9 turn. Thus, this finding is consistent with the interpretation that the 4/5 sequence introduced into the 8/9 turn did not result in a conversion to the 4/5 turn conformation. Although we were unable to obtain structural data for the S50E/V51N/G52H triple mutant, the available data show that swapping the amino acid sequence of the 4/5 and 8/9 turns does not concomitantly convert the turn conformations. Therefore, we conclude that the local environment influences both the stability and the conformation of the 4/5 and 8/9 turns.
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groups) (Hutchinson and Thornton 1994; Kwasigroch et al. 1996; Takano et al. 2001; Kim et al. 2003). The S50G/V51G double mutant and E91G/N92G/H93G triple mutant both resulted in a modest stabilization of the protein (–1.3 and –2.6 kJ/mol, respectively). Thus, a poly-glycine sequence in both turns can effectively substitute for the wild-type sequence (position 52 in the 4/5 turn is glycine in the wild-type protein). However, despite the similar overall stability effects of such a substitution, there appear to be notable differences in these turn regions with regard to structural strain.
A comparison of the wild-type protein with the S50G/V51G double mutant, and similarly, a comparison of the H93G point mutant with the E91G/N92G/H93G triple mutant, evaluates the effect of glycine mutations at the i+1 and i+2 positions within the 4/5 and 8/9 turns when the i+3 position is a glycine. In the case of the 4/5 turn, the conversion of the i+1 and i+2 positions to glycine results in a modest stabilization of –1.3 kJ/mol, and does not result in any structural changes within the turn region (Fig. 4). In the case of the 8/9 turn, the conversion of the i+1 and i+2 positions to glycine results in a significant destabilization of +4.0 kJ/mol (Table 1). A comparison of the H93G and G52H point mutations (i.e., comparing the effects of glycine vs. histidine at the i+3 position within the 4/5 and 8/9 turn regions) yields relatively consistent results for both turns, namely a destabilization of 5.5 to 6.6 kJ/mol upon the introduction of a histidine for glycine at this position. Thus, the 8/9 turn i+3 histidine exhibits significant structural strain (being located in the left-handed 
-helical region of the Ramachandran plot) (Kim et al. 2002), whereas the glycine residue at this symmetry-related position in the 4/5 turn does not. Conversely, the i+1 and i+2 residues in the 8/9 turn actively contribute to the stability of the structure (which is destabilized when converted to glycine), whereas similar positions in the 4/5 turn do not. These results identify a fundamental difference between these two turns: the 4/5 turn appears generally optimized and to have limited strain associated with it, whereas the 8/9 turn exhibits an inherent structural strain, and can be further optimized to substantially (i.e., ~6.6 kJ/mol) increase protein stability. Thus, while a simple polyglycine substitution within both turn regions can be accommodated, additional stability is achievable with an appropriate selection of side chains (e.g., E91/N92/G93 in the 8/9 turn region).
What structural features outside of the immediate turns might be responsible for influencing the structure of the turns? An examination of an overlay of the 4/5 and 8/9 turn regions provides some possible answers. If the 8/9 turn adopted the 4/5 turn structure, it would result in a 2.9 Å close contact between the main chain carbonyl oxygen of position E91 (equivalent to S50) and the side chain L89 C
1 atom (Fig. 5). Associated with this contact is the hydrogen-bonding requirement of the main chain carbonyl group of E91, which could not be satisfied by such an interaction; thus, this conformation appears unfavorable. A more egregious close contact would occur if the 4/5 turn adopted the 8/9 turn structure. In this case an ~0.5 Å contact would occur between a side chain C
atom of V51 (equivalent to N92) and the side chain O
1 atom of E53 (which is involved in a salt bridge with adjacent K100) (Fig. 5). The above interactions involve residues in the primary structure that are adjacent to the turn residues (i.e., positions 48/89 and 53/94); thus, the conformation of the residues within the turns appears to be determined not by the primary structure of the turn residues, but rather by the residues flanking the turns. In this regard, the main chain atom overlay of the two turns (Fig. 2) indicates that the structural deviations initiate with the angles of residue positions 49 and 90, and extend through the angles of residues 53 and 94. These different main chain , angles may dictate the observed structural difference within the two turns; however, since the residues at these positions are neither proline or glycine, the differences in main chain angles must be due to side chain interactions. Again, the results suggest that such interactions do not involve the side chains within the immediate turn region.
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-hairpin structures in proteins, the principles underlying their formation and stability are not well understood. There are two different views regarding the contributions of residues within turn regions upon conformation. One view is that turn residues can determine the turn conformation (de Alba et al. 1997). The other view is that turn regions in a protein may be able to maintain their conformation despite variations in their primary structure (Searle et al. 1995). Several groups have examined the influence of turn sequences on the formation and stability of -hairpins (Ramirez-Alvarado et al. 1997; Blanco et al. 1998; de Alba et al. 1999b; Griffiths-Jones et al. 1999; Chen et al. 2001). However, these studies used relatively small peptides, and the effects of adjacent regions outside of the turn (as would be present in larger globular proteins) could not be determined. In the present study, involving two symmetry-related turns within FGF-1, the data indicate that neither glycine substitution, nor sequence swapping, affected the local turn conformation. Furthermore, structural analysis suggests a role for flanking residues in determination of the turn conformation. These results support the general hypothesis of Searle et al. (1995) with regard to turn sequences and structures within proteins. Thus, predicting the stability and structure of turn regions within proteins requires knowledge of the larger structural context. However, the results suggest that a simple polyglycine primary structure will suffice for mesophilic applications. Finally, the results show that symmetry-related turn regions within a symmetric superfold can exhibit substantially different properties with regard to strain and contributions to overall folding kinetics. |
Materials and methods |
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Isothermal equilibration denaturation
Protein samples (40–10 µM) in various concentrations of GuHCl/ADA buffer were allowed to equilibrate overnight at room temperature (298 K). This study made use of the fluorescence signal of the single endogenous tryptophan residue at position 107. This residue is ~90% buried in the native structure (Blaber et al. 1996). Complete details of the instrumentation, data collection, and analysis procedure were reported (Blaber et al. 1999). Briefly, the fluorescence signal of FGF-1 is atypical in that W107 exhibits greater quenching in the native state rather than the denatured state. Excitation at 295 nm provides selective excitation of W107 in comparison with the six tyrosine residues that are present in the structure (Blaber et al. 1999; Brych et al. 2001). Triplicate scans were collected and averaged, and buffer traces were collected and subsequently subtracted from the protein scans. All scans were integrated to quantify the total fluorescence as a function of denaturant concentration. The data were analyzed using the general purpose nonlinear least-squares fitting program DataFit (Oakdale Engineering) implementing a six-parameter, two-state model (Eftink 1994):
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where the denaturant concentration is given by [D]; the native state (0 M denaturant) fluorescence intercept and slope are F0N and SN, respectively; the denatured state fluorescence intercept and slope are F0D and SD, respectively; and the free energy of unfolding function intercept and slope are G0 and m, respectively. The G0 and m values describe the linear function of the free energy of unfolding as a function of denaturant under isothermal equilibrium conditions (where G0 is the G value extrapolated to 0 M denaturant, and m is reported as –dG/d[D]). The midpoint of the transition, i.e., the denaturant concentration where G = 0, is defined as Cm. The effect of a given mutation upon the stability of the protein ( G) was calculated using the method of Pace (Pace and Scholtz 1997):
 | (2) |
where a negative value indicates that the mutation is stabilizing in relationship to the wild-type protein.
Folding kinetic measurements
Denatured protein samples were prepared by overnight dialysis against 20 mM ADA, 100 mM NaCl, 2 mM DTT (pH 6.60) containing either 2.5 M or 3.0 M GuHCl (Blaber et al. 1999). Initial studies using manual mixing indicated that the relaxation times for the folding process were more appropriate for stopped-flow data collection. All folding kinetic data were collected using a Kintek SF2000 stopped-flow system (Kintek). Folding was initiated by a 1:10 dilution of 40 µM denatured protein into 20 mM ADA, 100 mM NaCl (pH 6.60) with denaturant concentrations varying from 0.25 M GuHCl, in increments of 0.05 M, to the midpoint of denaturation as determined by the above described isothermal equilibrium denaturation measurements. The data collection strategy was designed to span approximately five half-lives, or >97% of the expected fluorescence signal change between the fully denatured and native states.
Unfolding kinetic measurements
Native protein samples were dialyzed against 20 mM ADA 100 mM NaCl, 2 mM DTT (pH 6.60) overnight at 298 K. Unfolding was initiated by a 1:10 dilution of 25 µM protein into 20 mM ADA, 100 mM NaCl (pH 6.60) with concentrations of GuHCl varying between 1.5 M and 5 M. The fluorescence signal associated with protein unfolding was quantified using a Varian Eclipse fluorescence spectrophotometer, with a wavelength of 295 nm for excitation and 350 nm for emission, and maintained at 298 K with a temperature-controlled Peltier cell holder (Varian). The unfolding kinetics exhibited relaxation times that were appropriate for manual mixing techniques. The data collection strategy was designed to span approximately 2–3 half-lives, or >80% of the expected fluorescence signal change between the native and denatured states.
Kinetic analysis
Both folding and unfolding kinetic data were collected in triplicate at each denaturant concentration, with typically six runs per sample and all data being averaged. The kinetic rate constants and amplitudes, as a function of denaturant concentration, were determined from the time-dependent change in fluorescence intensity implementing a single exponential model:
 | (3) |
where I(t) is the intensity of fluorescent signal at time t, A is the corresponding amplitude, k is the observed rate constant for the reaction, and C is a constant corresponding to the asymptotic signal limit.
Folding and unfolding rate constant data were fit to a global function describing the contribution of both rate constants to the observed kinetics as a function of denaturant ("Chevron" plot) as described by Fersht (1999):
 | (4) |
where kf0 and ku0 are the folding and unfolding rate constants, respectively, extrapolated to 0 M denaturant; mkf and mku are the slopes of the linear functions relating ln(kf) and ln(ku), respectively, to denaturant concentration; and D is denaturant concentration.
Crystallization of FGF-1
Purified FGF-1 solutions (~13 mg/mL) were filtered through 0.2- µm filters (Pall Life Sciences) immediately prior to crystallization trials. X-ray diffraction quality crystals of S50G/V51G grew within 1 wk at RT in 10- µL hanging drops by vapor diffusion against 1-mL reservoirs of 3.0–3.2 M formate and 0.9 M ammonium sulfate in crystallization buffer. V51N and S50E/V51N crystals were obtained within 1 wk at RT in 10- µL hanging drops by vapor diffusion against 3.4–3.6 M formate.
Data collection, molecular replacement, and refinement
X-ray diffraction data were collected using a Rigaku RU-H2R rotating anode X-ray source (Rigaku) equipped with Osmic Blue, or Purple, confocal mirrors (MarUSA) coupled with either a Rigaku R-axis IIc image plate or a MarCCD 165 detector. The crystals were mounted using Hampton Research nylon-mounted cryo-turns and frozen in a stream of nitrogen gas at 100K. Diffraction data were indexed, integrated, and scaled using the DENZO software package (Otwinowski 1993; Otwinowski and Minor 1997). His-tagged wild-type FGF-1 was used as the search model in molecular replacement in each case. Model refinement was carried out with CNS (Brunger et al. 1998) using the maximum likelihood target function. Model building and visualization utilized the O molecular graphics program (Jones et al. 1991).
NMR studies
NMR samples of FGF-1 in 100 mM phosphate buffer, 200 mM ammonium sulfate, and 2 mM DTT (pH 6.0), were labeled uniformly with 15N or 13C using conditions described by Chi et al. (2002). The protein was concentrated to 1 mM, dried by lyophilization, and then resuspended by dissolving in 10% D2O containing 10 mM phosphate buffer with 90% H2O. The two- and three-dimensional NMR experiments were performed at 30°C on a 600 MHz Varian UnityPlus spectrometer equipped with waveform generators and pulsed field gradient accessories. Individual spin systems of the wild-type protein were identified via TROSY experiments and then confirmed by 3D HNCACB and CBCA(CO)NH. Individual spin systems of mutant proteins were assigned by HSQC and 3D 15N TOCSY-HSQC experiment.
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Acknowledgments |
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References |
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