1Laboratoire de Modélisation et dIngénierie des Protéines, UMR 8619, Université de Paris-Sud, Bât. 430 and 2Laboratoire de Biophysique Moléculaire, INSERM U 350, Institut Curie, Université de Paris-Sud, Bât 110, F-91405 Orsay Cedex, France M.Nicaise and M.Valerio-Lepiniec contributed equally to this work.
3 To whom correspondence should be addressed. e-mail: michel.desmadril{at}mip.u-psud.fr
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Abstract |
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Keywords: ß-sheet protein/immunoglobulin fold/neocarzinostatin/tyrosine corner
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Introduction |
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Various studies have shown that apoNCS can bind other natural chromophores (Kappen et al., 1980) or small-molecule drugs (Urbaniak et al., 2002
). The ability of apoNCS to bind to other small-molecule drugs may offer opportunities for delivering other entities in vivo. However, such applications would depend not only on the ability of apoNCS to accommodate molecules other than natural chromophores within the binding cleft but also on the ability to target the drug. Before using protein engineering to remodel the loops for drug targeting, we need to know which are the main stabilizing interactions that should not be altered.
In a broader perspective, the three-dimensional structure of neocarzinostatin is based on a seven-stranded antiparallel ß-sandwich, very similar to the immunoglobulin folding domain (Adjadj et al., 1992a,b; Kim et al., 1993
). Although there is a large amount of data concerning the stability and folding of various immunoglobulin domain proteins (Clarke et al., 1999
; Lorch et al., 1999
; Fowler et al., 2002
) and also about the structural determinants in the sequences of immunoglobulin variable domains (Chothia et al., 1998
), few data are available for NCS or other members of this family. As NCS and the other proteins of the immunoglobin fold family have no sequential or functional link, it is important to determine the extent to which their structural properties are similar and, conversely, the implications of differences in structural features for maintaining the stability of the fold. The similarity between the NCS and the immunoglobulin fold is clearly shown in the topological diagram in Figure 1. The first sheet of both ß-sandwiches includes A, B and E strands and the second sheet consists of D, C, F and G strands.
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Materials and methods |
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The cloning of the neocarzinostatin gene has been described previously (Heyd et al., 2000). Mutagenesis was performed using the Stratagene Quickchange kit. Point mutation was identified using restriction digestion (silent removal site) and DNA sequencing of complete NCS DNA. The 6x His-tagged protein was over-produced in Escherichia coli BLR21 (Novagen) and then purified on an Ni-NTA matrix (Qiagen). All experiments were performed in 20 mM phosphate buffer, pH 5.4.
Physicochemical properties
Circular dichroism (CD) spectra were recorded from 185 to 250 nm on a JASCO dichrograph equipped with a thermostatically controlled cell holder and connected to a computer for data acquisition. Data were acquired from 13 µM sample solutions in quartz cells of 1 mm pathlength.
All NMR spectra were acquired on a Varian Unity 500 spectrometer under the conditions described elsewhere (Adjadj et al., 1990, 1991).
Ethidium bromide (EtBr) binding to WT and mutant apo-NCS was studied by fluorimetry with an Aminco SLM 8000 fluorimeter by monitoring the intrinsic fluorescence of a EtBr solution (5 µM final concentration, exc = 479 nm,
em = 620 nm, bandwidth 2 nm) at various apo-NCS concentrations. Saturation curve data were analyzed by using the following equation:
where Fmax = Fmax F0,
F = F F0, F and F0 are the fluorescence intensity measured in the presence and absence of the protein, respectively, P0 is the total protein concentration, B0 is the total ethidium bromide concentration and Kd is the dissociation constant. Experimental data were fitted according to Equation 1 by using a simplex procedure based on the Nelder and Mead algorithm (Heyd et al., 2000
).
Stability of apo-NCS WT and mutant
Thermal stability was studied by differential scanning calorimetry (DSC) on a Microcal instrument with a 1 mg/ml apo-NCS solution dialyzed overnight against standard phosphate buffer. Buffer solution from the dialysis bath was used as a reference. All solutions were degassed just before loading into the calorimeter. Scanning was performed at 1 K/min. Reversibility of the denaturation process was checked by re-scanning a denatured sample after cooling.
The heat capacity of the solvent alone was subtracted from that of the protein sample. These corrected data were analyzed using a cubic spline as a baseline in the transition. Thermodynamic parameters Hcal and
Hvh were determined by fitting the following equation to the data:
where Kd is the equilibrium constant for a two-state process and Hcal is the measured enthalpy, corresponding to
and Hvh is the enthalpy calculated on the basis of a two-state process. If the measured transition corresponds to a two-state process, the values of the two enthalpies
Hcal and
HvH are equal. Values of the ratio
Hcal/
Hvh other than 1 imply the presence of intermediates or multimolecular processes (Freire, 1995
).
For spectroscopic measurements, the fraction unfolded fu was calculated from the signal using the standard equation
where N and
D represent, respectively, the signal value of the native and denatured species at each temperature, taking into account the baselines preceding and following the transition region.
HvH and T1/2 were calculated from the transition curves using the standard equation
where K is defined as
and HvH is assumed to be temperature independent and R is the gas constant. Tm was estimated for lnK = 0.
The unfolding induced by guanidinium chloride (GdmCl) of 5 µM protein solutions in standard buffer was monitored by fluorescence spectroscopy. Fluorescence was measured with an Aminco SLM 8000 fluorimeter, after 12 h of incubation at 4°C in solutions of various GdmCl concentrations. Ultra-pure GdmCl was obtained from Pierce; the denaturant concentrations were checked by refractometry, using the relationship provided by Nozaki (Nozaki, 1970). The transition curves were constructed by plotting the position of maximum fluorescence emission (
exc = 295 nm, bandwidth 2 nm) against denaturant concentration.
The model of the linear dependence of Gx upon denaturant concentration, x, according to Pace (Pace, 1986
) was used for thermodynamic analysis:
Assuming that the linear dependence of the free energy change on denaturant concentration observed in the transition region can be extrapolated to zero denaturant concentration, G0 represents the standard variation of free energy in the absence of denaturant and m is a constant proportional to the increase in the accessible surface area of the protein to the solvent on denaturation. An equation derived from Equation 7, taking into account the baselines and the transition region, was used to analyze the data:
where yx is the experimental signal in the presence of x M GdmCl, yn is the signal of the native form, sn and sd are the solvent effects on the native and denatured protein signal, respectively, and A is the amplitude of the transition. Experimental data were fitted according to Equation 8 by using a simplex procedure based on the Nelder and Mead algorithm.
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Results |
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The overall structure of the mutant was evaluated by comparing the CD spectra of the mutant and the wild-type protein (Figure 3). The spectra are fully superimposable and have a signal characteristic of an all-ß-protein with a maximum at 195 nm and a minimum at 210 nm. The mutant Y32F spectrum also displays a positive contribution at 223 nm, which is specific to the apo-NCS. This maximum has been proposed to result from constraints in the polypeptide backbone and representing no typical secondary structure (Heyd et al., 2000
).
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Integrity of the binding site
The integrity of the cleft was checked by measuring EtBr binding. This compound binds apo-NCS stoichiometrically in the natural chromophore cleft (Mohanty et al., 1994) and this is therefore a convenient tool for monitoring the functional properties of apo-NCS and its variants. The Kd value obtained at pH 5.4 for the Y32F mutant (29 µM) is different from that of the wild-type protein (16 µM) determined under the same conditions. The slight but significant increase in Kd indicates that this mutation induces changes in the conformation and/or dynamic properties of the binding site of the EtBr.
Stability of the mutant
Thermal stability of the mutant Y32F followed by DSC.
The thermal stabilities of mutant Y32F and wild-type NCS were compared using DSC (Figure 4). The transition was fitted with a non-two-state model. At pH 5.4, the midpoint temperature of the transition for the Y32F mutant is Tm = 60.3 ± 0.4 versus 69.3 ± 0.4°C for the wild-type. The calorimetric enthalpy of the mutant (Hcal = 94.5 ± 1.1 kcal/mol) was lower than that of the wild-type (111.7 ± 1.1 kcal/mol). The
HvH/
Hcal ratio for both proteins was close to one (1.05 for the Y32F and 0.96 for the WT). These results suggest that the Y32F mutation has significant effects on the overall stability of the protein.
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Discussion |
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The substitution YF does not change the overall structural features of the protein. At 25°C the fluorescence properties of the mutant are comparable to those of the WT, suggesting that the mutation does not significantly modify the accessibility of W39, the main contributor to fluorescence. The CD spectrum of the mutant protein is identical with that of the WT protein, indicating that the mutation does not change the overall secondary structure content of the protein. Hence the observed changes in protein stability are mainly related to changes in local interactions.
Effects of the mutation on protein stability
Removing the tyrosine hydroxyl moiety reduces the stability of NCS as assessed by microcalorimetry and denaturation monitored by CD and fluorescence. DSC measurements indicate that the Tm of the Y32F mutant is 9°C lower than that of the wild-type protein; this shift in Tm is accompanied by a decrease of 16 kcal/mol in
Hcal. This decrease in stability corresponds also to a change in the denaturation process. Thermal denaturation monitored by fluorescence clearly shows a double transition, the first one with a Tm corresponding to that determined by microcalorimetry and the second with a higher Tm, corresponding to the denaturation of a more stable local residual structure.
The substitution of tyrosine by phenylalanine leads to the occurrence of intermediate states which were not detected in the wild-type. This has been observed for apo-pseudoazurin, another protein with a 5 tyrosine corner, in which intermediate states were observed after substituting tyrosine with tryptophan (Jones et al., 2000
). It must be emphasized that NCS does not undergo a true two-state heat denaturation process and presents an unexpected complex unfolding transition with temperature (Perez et al., 2001
). Hence the presence of two transitions could simply be related to a change in the relative stability of one or more intermediates that have been detected by SAXS experiments (Perez et al., 2001
).
CD and NMR measurements show that both secondary structure and overall folding are identical in mutant Y32F and wild-type protein. However, some chemical shift variations were observed for residues in contact with the tyrosine corner. Remote effects are observed at the base of the ß-sandwich and in particular for residues 7071 and 88. In addition, the integrity of the ligand-binding site is affected by the substitution of tyrosine. The value of the dissociation constant obtained for mutant Y32F is about twice as high as that obtained for the wild-type. The structure of the mutant is similar to, if not identical with, that of the wild-type and therefore the slightly reduced affinity for EtBr of mutant Y32F is probably due to a weakening of attractive interactions between the protein and ligand, which could be due to an increase in the internal flexibility of the protein. Indeed, it has been shown that Y32 takes an important part in maintaining the rigidity of the top of the ß-sandwich (Izadi-pruneyre et al., 2001). Removing the OH-bond could release this internal constraint. This hypothesis is consistent with change in the m values for the mutant Y32F, characteristic of an increase in the mean accessible surface area of the native protein.
Comparison with other tyrosine corners
The substitution Y32F has various effects on the stability of the Ig fold. Hamil et al. have worked on various proteins of the fibronectin type III superfamily (Hamil et al., 2000). They have shown that the mutation Y
F costs between 1.5 and 3 kcal/mol. In some cases, the destabilization induced by this replacement is so large that the protein cannot fold, as is the case for pseudoazurin (Hazes and Hol, 1992
). Although for NCS the destabilization is fairly large (4 kcal/mol), this mutant is able to acquire a native fold. This raises the question as to why the tyrosine corner motif is found ubiquitously and exclusively in Greek key proteins and why the hydrogen bond is necessary for their stability. It has been shown that the tyrosine corner is not a folding nucleus and that this structure has mainly a stabilizing role (Hamill et al., 2000
). Indeed, the diverse effects of the Y
F substitution in closely related local structures suggest that, as observed for other interactions such as N- and C-cap, the environment can modulate the stabilizing role of the hydrogen bond. The pseudoazurin tyrosine corner consolidates a ß-zipper structure between strands VI and VII and is thus essential to the protein stability (Hazes and Hol, 1992
; Jones et al., 2000
). In NCS, although the Tyr corner is important for the stability of the protein, it is not essential. This could be related to the localization of the Tyr corner in this protein. In NCS, the Tyr corner is not localized on the EF loop, where it is observed for the majority of Ig fold proteins, but on the BC loop. In this protein, the stabilization of the EF loop is obtained via a disulfide bridge (Cys88Cys93) that is essential for NCS stability, its elimination being lethal for the protein. As described in a study of the dynamic properties of apo-NCS (Izadi-pruneyre et al., 2001
), the tyrosine corner allows a structural cohesion with the other side of a protein. Substitution with phenylalanine induces greater flexibility of the loop and this is tolerated in the protein structure.
Although our data do not rule out the possibility that the tyrosine corner could also play a role in the folding process, we suggest that the differences between the role of the tyrosine corners in related proteins may originate from their specific functions. For immunoglobulins, the BC loop (CDR1) is stabilized by a disulfide bridge, providing the rigidity required for correct recognition of the antigen. In contrast, for NCS, the tyrosine corner has a stabilizing role in the same loop, but also plays a role in the correlated internal movements needed for correct ligand binding. With a view to using this protein for drug targeting, these results, combined with those obtained previously (Valerio-Lepiniec et al., 2002), allow us to define clearly the limitations of the modifications that can be performed on this scaffold.
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Acknowledgements |
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Received December 3, 2002; revised June 6, 2003; accepted September 3, 2003.