Key interactions in neocarzinostatin, a protein of the immunoglobulin fold family

Marielle Valerio-Lepiniec1, Magali Nicaise1, Elisabeth Adjadj2, Philippe Minard1 and Michel Desmadril1,3

1 Laboratoire de Modélisation et d’Ingénierie des Protéines, UMR8619, Université de Paris-Sud, Bât 430, F-91405 Orsay Cedex and 2 Laboratoire de Biophysique Moléculaire, INSERM U 350, Institut CurieUniversité de Paris-Sud, Bât 110, F-91405 Orsay Cedex, France


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
Neocarzinostatin (NCS) is a seven-stranded ß-sandwich protein, the folding of which is similar to that of the variable domains of immunoglobulins (Ig). The investigation of the backbone dynamics of apo-NCS [Izadi-Pruneyre et al.(2001)Go Protein Sci., 10, 2228–2240] enabled us to identify the involvement of long side-chain residues in maintaining the rigidity of this ß-protein. In the perspective of using this protein for drug targeting, this raises the following question: do these residues also play a key role in the stabilization of the ß-sheet? To investigate this problem, various genetically engineered variants were constructed by mutating these residues to amino acids with shorter aliphatic side chains. These substitutions have no effects on the global fold. However, an important destabilization of the protein, higher than that expected for a simple `large-to-small’ substitution of buried hydrophobic residues, is observed for three mutants, V34A, V21A and V95A. Interestingly, the nature of the residues in these positions is highly conserved in the other Ig-like proteins. The absence of an evolutionary relationship between NCS and the other Ig-like proteins strongly suggests that this hydrophobic core is characteristic of the Ig-fold itself.

Keywords: hydrophobic interactions/immunoglobulin fold/neocarzinostatin


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
It is well known that the folded conformation adopted by a protein is determined by its amino acid sequence; however, we still cannot predict the three-dimensional structure of a protein from its sequence alone. What makes this prediction so difficult is that the folded state is unique within a vast number of possible conformations. The problem could be simplified by deciphering the rules underlying the folding process. To achieve this, we must determine the relative importance of different contributions to protein stability and elucidate the role of these contributions in protein folding. These contributions include the hydrophobic effect, hydrogen bonding, steric hindrance and electrostatic interactions. The hydrophobic effect was first considered as the main driving force for protein folding, as it was simply considered in terms of the favorable entropy associated with the transfer of non-polar amino acids from water into the predominantly non-polar medium within the protein (Kauzmann, 1959Go, 1987Go; Privalov and Gill,, 1988Go; Dill, 1990Go). With the arrival of protein engineering and the use of statistical analysis of protein databases, more precise information about the role of hydrophobic interactions has been obtained. In particular, detailed studies on a variety of proteins have shown that the detailed packing of non-polar atoms and the burial of the hydrophobic surface area are very important in the stabilization of proteins (Dill, 1990Go; Sturtevant, 1994Go; Matthews, 1995Go).

Neocarzinostatin (NCS), secreted by Streptomyces neocarzinostaticus, is the best known of a family of macromolecular chromoprotein antibiotics which also have antitumoral activity. The other members are macromomycin (Van Roey and Beerman, 1989Go), secreted by Streptomyces macromomyceticus, C-1027 (Xu et al., 1994Go) and actinoxanthin (Sakata et al., 1993Go), secreted by Streptomyces globisporus, maduropeptin (Zein et al., 1995Go) secreted by Actinomadura madurae and kedarcidin (Constantine et al., 1994Go) secreted by an unidentified species of actinomycetes.

NCS consists of a labile chromophore (NCS-Chr) (Mr = 659) (Edo et al., 1986Go) that is tightly and non-covalently bound to an apoprotein (apo-NCS) (113 amino acids) (Adjadj et al., 1992Go). The chromophore, with an unusual bicyclic dienediyne structure, is very potent in causing DNA damage. Although apo-NCS itself is inactive in inducing cleavage of DNA, it plays an important role in the drug action by protecting and storing the labile chromophore and by releasing the chromophore to the target DNA. The biologically active chromophore has a very high affinity for its apo-NCS (Kd ~ 10-10 M), the role of the apo-NCS being to stabilize this highly unstable component.

Although NCS is very potent against tumor cells, it damages normal cells as well. Thus, one goal of engineering NCS is to reduce this damage by tumor-targeting of the drug. This has already been tested either by chemical modification of the protein (Noda et al., 1990Go) or by covalent linkage to a murine monoclonal antibody A7 which recognizes the glycoprotein on the surface of human colon cancer cells (Kitamura et al., 1993Go). Another way to reduce the side effects described above is to use protein engineering to remodel the chromophore binding site in order to deliver other potent drugs having fewer secondary effects. However, before modifying the sequence, we need to know what are the main stabilizing interactions that should not be altered.

In a broader perspective, NCS also shares the same overall fold of that of an immunoglobulin (Ig) domain. Like other Ig-like proteins, apo-NCS has no disulfide bridge between the two ß-sheets, however, two other disulfide bridges are present, located in the cleft (C37–C47 for NCS) and at the bottom of the barrel (C88–C93 for NCS) (Adjadj et al., 1992Go).

Recently, NMR and molecular dynamics (Izadi-Pruneyre et al., 2001Go) enabled us to correlate the dynamics of this ß-protein with key amino acid interactions. In particular, this led to the identification of a hydrophobic cluster within the core of the apo-NCS ß-sandwich. To evaluate the implication of these residues in the stability of NCS, in the present work we replaced the amino acids at these positions by residues with shorter side chains, allowing us to decrease the hydrophobic interactions without inducing significant structural changes. Using a variety of approaches to study the structure and stability of these mutants, we show that three positions seem to be essential for the stabilization of the ß-sandwich. This gives an important contribution to our understanding of the forces that stabilize ß-sandwich folded proteins and can aid in the construction of a genetically modified protein suitable for drug targeting.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
Mutagenesis and protein purification

NCS was cloned as previously described (Heyd et al., 2000Go). In this construct, the coding sequence is fused to the ompT signal sequence, to direct the secretion of the protein into the periplasm. The Stratagene Quickchange kit was used for mutagenesis. Point mutations were identified by restriction digestion (silent addition or removal of sites) and by DNA sequencing of the entire NCS gene. Mutant plasmids were transformed into Escherichia coli BLR21 (Novagen). Proteins were produced and purified as described previously (Heyd et al., 2000Go).

Physicochemical properties

The molecular weight and the purity of the variant protein was analyzed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry using standard methods.

Circular dichroism (CD) spectra were recorded from 185 to 250 nm on a Mark VI dichrograph (Jobin-Yvon) equipped with a thermostatically-controlled cell holder and connected to a computer for data acquisition. Data were acquired from 13 µM samples in 20 mM phosphate buffer, pH 5, in quartz cells with a 1 mm path length.

Ethidium bromide (EtBr) binding was studied at 25°C and pH 5.4, by fluorimetry with an Aminco SLM 8000 fluorimeter by monitoring the intrinsic fluorescence of an EtBr solution (5 µM final concentration, {lambda}exc = 479nm, {lambda}em = 620 nm, band width of 2 nm) as a function of apo-NCS concentration (Mohanty et al., 1994Go). The saturation curve data was analyzed by the following equation:

(1)
where {Delta}Fmax = Fmax F0, {Delta}F = FF0 and where 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 EtBr concentration and Kd is the dissociation constant. Experimental data were fitted according to Equation 1Go by a simplex procedure based on the Nelder and Mead algorithm (Press et al., 1986Go).

Stability of WT and mutants apo-NCS

Thermal stability was studied by differential scanning calorimetry (DSC) on a differential scanning calorimeter (Microcal). DSC measurements were made with a 1 mg/ml apo-NCS solution dialyzed overnight against 20 mM phosphate buffer, pH 5. The buffer 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. The percentage of recovery of the native protein after heat denaturation was evaluated by re-scanning after the denatured sample had cooled.

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, calorimetric enthalpy ({Delta}Hcal) and van’t Hoff enthalpy ({Delta}HvH), were determined by fitting the following equation to the data:

(2)
where Kd is the equilibrium constant for a two-state process, {Delta}Hcal is the measured enthalpy, corresponding to:

(3)
and {Delta}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 {Delta}Hcal and {Delta}HvH, are equal. Values for the ratio {Delta}Hcal / {Delta}Hvh other than 1 imply the presence of intermediates or a multimolecular process (Freire, 1995Go).

For spectroscopic measurements, the unfolded fraction fu was calculated from the signal {Theta} using the standard equation:

(4)
where {Theta}N and {Theta}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. {Delta}HvH and the midpoint transition temperature (Tm) were calculated from the transition curves using the standard equation:

(5)
where K is defined as:

(6)
and {Delta}HvH is assumed to be temperature-independent and R is the gas constant. Tm was estimated for ln K = 0.

The unfolding experiments induced by guanidinium chloride (GdmCl) were monitored by fluorescence spectroscopy on 5 µM protein solutions in 20 mM phosphate buffer, pH 5. The fluorescence was measured with an Aminco SLM 8000 fluorimeter, after 12h of incubation at 4°C in various concentrations of GdmCl. Ultra-pure GdmCl was obtained from Pierce; the denaturant concentrations were checked by refractometry, using the relationship provided by Nozaki (Nozaki, 1972Go). The transition curves were constructed by plotting the position of maximum fluorescence emission ({lambda}exc= 290 nm, band width =2 nm) as a function of denaturant concentration.

Thermodynamic analysis was performed by use of the model of linear dependence of {Delta}Gx upon denaturant concentration, x, according to Pace (Pace, 1986Go):

(7)

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, {Delta}G0 represents the standard change of free energy in the absence of the denaturant and m is a constant proportional to the increase in the accessible surface area of the protein to the solvent on denaturation. The data were analyzed by use of an equation derived from equation (7Go), taking into account the baselines and the transition region:

(8)
where yx is the experimental signal in the presence of x molar 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 (8Go) by use of a simplex procedure based on the Nelder and Mead algorithm (Press et al., 1986Go).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
Mutational sites

The strong structural similarity between apo-NCS and members of the Ig-fold super-family raises questions about the nature of the stabilizing forces between the two ß-sheets because these proteins do not share discernible sequence identity. We used two kinds of information to determine which interactions are likely to be the most important for the stability of NCS.

First, we compared the sequence of apo-NCS with the other members of the Ig-fold superfamily to detect possible related patterns of hydrophobic interactions. Sequence alignment between apo-NCS and fibronectin was performed on a topological basis, using the TOP program (Lu, 2000Go), and this alignment was compared to other alignments obtained from literature (Table IGo). This revealed that all of these Ig-like proteins have the same pattern of hydrophobic interactions even though there is no significant residue identity. This pattern corresponds to that observed by Hamill et al. (Hamill et al., 2000Go). These authors compared the folding properties of the Ig-like ß-sandwich proteins and reported that this folding is mainly initiated by a stabilizing packed unit of structures consisting of hydrophobic residues in the central B, C, E and F strands (Hamill et al., 2000Go). For the third fnIII domain from human tenascin (TNfn3) (Leahy et al., 1992Go), one of these ß-sandwich proteins, this particular BCEF motif is typically characterized by residues: Ala18, Ile20, Leu34, Tyr36, Ile59, Tyr68, Val70 and Leu72 (Clarke et al., 1999Go). The sequence alignment based on the structural superimposition of apo-NCS with TNfn3 clearly shows that these two proteins display a similar pattern of interactions even though there is no significant residue identity. This well defined interaction packing suggests that they are involved in stabilizing the Ig-fold.


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Table I. Sequence alignment based on the structural superimposition of apo-NCS with TNfn3 created by use of the TOP program (Lu, 2000Go)
 
Secondly, we recently reported the analysis of the intramolecular motions of apo-NCS by use of NMR and molecular dynamics (Izadi-Pruneyre et al., 2001Go). This study led to the identification of a hydrophobic cluster within the core of the apo-NCS ß-sandwich, essentially formed from the residues of the BCEF strands: Val21, Val34, Gln36, Leu67, Val95 and Leu 97. Interestingly, these positions correspond to the pattern of interactions identified after sequence alignment based on the structural superimposition illustrated in Table IGo. Moreover, this pattern is similar to the hydrophobic cluster identified by NMR and molecular dynamics (Izadi-Pruneyre et al., 2001Go). Given that the X-ray structure showed that the hydrophobic residues V21, V34, L67, V95 and L97 are completely buried (Teplyakov et al., 1993Go), we chose to mutate these residues to investigate their stabilizing properties in apo-NCS. These residues of the core of the NCS are indicated on the three-dimensional structure of apo-NCS in Figure 1Go.



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Fig. 1. Hydrophobic residues in the core of NCS. The figure was prepared by use of the Swiss PDB viewer program, with the pdb file 1noa.pdb (Teplyakov et al., 1993Go). The residues mutated in this study (Val21 of strand B, Leu67 of strand E, Val34 of strand C, Val95 and Leu 97 of strand F) are shown in space filling.

 
Mutations were designed to decrease hydrophobic interactions avoiding significant structural changes. This was achieved by performing the following substitutions V21A, V34A, L67V, V95A and L97V.

Characterization of the mutants

For each mutant protein, an overloaded SDS–PAGE gel showed one well resolved band with no visible contamination. To ensure that the mutations did not induce significant change in the protein structure, the recombinant proteins were further characterized. We first checked that the mature products corresponded exactly to the encoded sequence. For each mutant, mass spectrometry indicated the presence of a single species with the expected molecular weight (data not shown). This indicates that the recombinant proteins were homogeneous and that the N- and C-terminal ends had not been subjected to proteolysis, except for the cleavage of the synthetic signal peptide (Heyd et al., 2000Go).

CD spectra were recorded for WT and mutant proteins under identical conditions at 25°C (Figure 2Go). All of the spectra are fully superimposable and had the characteristic signal of an all ß-protein with a maximum at 195 nm and minimum at 210 nm. The mutant spectra also showed the positive contribution at approximately 223 nm, which is specific to the apo-NCS and which has been reported as representing `no typical’ secondary structure (Perczel et al., 1996Go; Christopher et al., 2000Go). Thus, the overall structure of the protein was not modified by the various substitutions.



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Fig. 2. CD spectra of WT, V21A, V34A and V95A protein. The CD spectra of L67V and L97V mutants are fully superimposable with WT (data not shown).

 
The fluorescence emission spectra of mutated NCS have a maximum emission wavelength of 343 nm, which is equivalent to that of the wild-type (WT) (data not shown). NCS has two tryptophan residues, Trp39 and Trp83. As shown by selective oxidation with N-bromosuccinimide in the presence of the chromophore (Edo et al., 1991Go), only Trp39 contributes significantly to the fluorescence signal. The accessibility of this residue to solvent is clearly not affected by the various substitutions showing that the mutations did not induce any significant structural changes in the distal loop located at the bottom of the cleft.

The integrity of the cleft was also examined by measuring EtBr binding. It has previously been shown that EtBr binds to NCS at the natural chromophore site (Adjadj et al., 1992Go; Proba et al., 1997Go). This provides a convenient way of monitoring the functional integrity of the binding site of the protein. The Kd values obtained for all mutants are very close or indistinguishable to the value obtained for the WT protein. Only the L97V mutant showed slightly lower Kd (10 µM) versus WT protein (16 µM). This Kd value obtained for the WT protein at pH 5.4 is slightly higher than that obtained at pH 7.5 (Heyd et al., 2000Go)

All these data indicate that none of the various single mutations induced any change in the conformation or in the functional properties of the protein.

Stability of mutants compared to WT

The stability of the mutant proteins was evaluated by use of spectroscopy and calorimetry to analyze unfolding transitions induced by heat and chemical treatment.

DSC study of the thermal stability of the modified proteins. DSC was used to compare the thermal stability of the mutants to that of the WT NCS (Figure 3Go). The unfolding of the WT protein resulted in a transition peak centered at 69.3°C (Table IIGo). The transition peak was analyzed by a non-two-state single transition model. This gave a {Delta}HvH / {Delta}Hcal ratio of 0.96 with a calorimetric enthalpy {Delta}Hcal of 111 kcal mol-1, suggesting a two-state model. As for the WT protein, the {Delta}HvH / {Delta}Hcal ratio of the V34A, L67V and L97V mutants was near to one. For these mutants the midpoint transition temperatures are very close to that of the WT, their Tm values being 68.6, 67.7 and 67.2°C, respectively (Table IIGo).



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Fig. 3. DSC of the WT ({circ}) and mutated NCS V34A ({blacktriangleup}), L67V ({blacksquare}), L97V ({blacklozenge}), V21A ({circ}), V95A ({square}) recorded in the same experimental conditions.

 

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Table II. Thermodynamic parameters of the heat denaturation as monitored by DSC
 
In contrast, the V21A and V95A mutations have significant effects on the overall stability of the protein. A decrease in the melting temperature is observed, with a shift of approximately 9 and 7°C for mutants V21A and V95A, respectively. The V95A mutation induces greater changes in the stability of the protein, the shift in melting temperature being accompanied by a decrease in denaturation enthalpy. Moreover, the {Delta}HvH / {Delta}Hcal ratio is <1 for both of theses mutant proteins, indicating a change in the overall folding process.

A second scan of each sample performed after cooling showed that we recovered >80% of the enthalpy for most of the mutant proteins, except the V95A mutant.

The results obtained by calorimetry suggested that positions 34, 67 and 97 do not play a major role in the thermal stability of NCS. Conversely, the stability was reduced significantly when positions 21 or 95 were affected.

Thermal denaturation of the proteins monitored by fluorescence. To obtain further information about the role of these positions on protein stability, the thermal transition was also studied by monitoring the variation of the maximum fluorescence emission wavelength of NCS and the mutant proteins (Figure 4Go). Fluorescence spectra of the WT and mutant proteins obtained by excitation at 295 nm were recorded at a series of temperatures between 25 and 85 or 90°C depending on the mutant. At low temperatures, the maximum emission wavelength was 343 nm, whereas at high temperatures it was 347 nm. This indicates that at high temperature Trp residues are largely exposed to the solvent. However, the exposition was not complete for any of the proteins studied, the maximum emission wavelength reaching only a value of 347 nm. This suggests that residual structures are present in the thermal denatured state. We report in Figure 4Go the variations of the maximum fluorescence emission wavelength normalized as a function of temperature for WT and mutants. The Tm is 71°C for the WT NCS, a very close Tm being obtained for mutant proteins V34A, L67V and L97V (Table IIIGo). This is fully consistent with the DSC results, suggesting that mutations in these positions do not affect significantly the thermal stability of the protein.



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Fig. 4. Unfolding transition curve assessed by the normalized variation of the maximum fluorescence intensity wavelength as a function of the temperature. WT (•) and mutated NCS V34A ({blacktriangleup}), L67V ({blacksquare}), L97V ({blacklozenge}), V21A ({circ}), V95A ({square}) have been recorded in the same experimental conditions.

 

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Table III. Analysis of heat denaturation as monitored by tryptophan fluorescence
 
The Tm for the V21A and V95A mutants, are 61 and 62°C, respectively. These findings further support the DSC results and confirm that the stability of NCS is significantly reduced when residues 21 or 95 are mutated. The curve obtained for mutant V21A displays two well resolved transitions, one centered at 59°C (Tm1) and another at approximately 70°C (Tm2) (Figure 4Go). Tm1 is consistent with DSC measurements, whereas the existence of a second Tm2 suggests that some local motifs of the protein have different stability.

Fluorescence study of the chemical denaturation of the proteins. We also studied the effects of denaturing WT and mutant NCS with GdmCl. Figure 5Go shows the normalized variation of the maximum-emission fluorescence wavelength of NCS and mutant proteins as a function of the GdmCl concentration.



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Fig. 5. Unfolding transition curve assessed by the normalized variation of the maximum fluorescence wavelength as a function of the GdmCl concentration. Comparison of the WT (•) and the single mutants V34A ({blacktriangleup}), L67V ({blacksquare}), L97V ({blacklozenge}), V21A ({circ}), V95A ({square}) recorded in the same experimental conditions.

 
The transition for the WT protein occurred at between 2 and 4 M GdmCl with a midpoint transition (Cm) of 3.02 M (Table IVGo). The calculated Gibbs free energy of unfolding ({Delta}G0) and the proportionality constant m in Equation (7Go) are 9.66 kcal mol-1 and 3.2 kcal mol-1 M-1, respectively.


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Table IV. Analysis of GdmCl denaturation as monitored by tryptophan fluorescence
 
The transition is complete at 4 M GdmCl. However, the fluorescence amplitude of the denaturation transition differs according to the denaturing agent. Thermal denaturation of the NCS leads to a protein with the maximum emission wavelength of approximately 347 nm, whereas when the protein is denatured by 4 M GdmCl it displays a maximum emission wavelength of approximately 349 nm, corresponding to fully exposed tryptophan residues.

Thermodynamic analysis of the transition of L67V and L97V mutants reveals a decrease of {Delta}G0 of approximately 0.4 kcal mol-1 for L67V and 1 kcal mol-1 for L97V. This small variation of {Delta}G0 is accompanied by a moderate shift in the Cm value without any change in the cooperativity of the process. The transition midpoint is 2.76 and 2.53 M for L67V and L97V mutants, respectively, and the m value is 3.3 kcal mol-1 M-1 for both mutants.

The {Delta}G0 value of V34A is 7 kcal M-1, which is lower than that of the WT. However, this lower stability is mainly due to the decrease in the m value (1.8 kcal mol-1 M-1), as the transition midpoint is shifted towards a higher denaturant concentration (3.87 M).

The transition of the V21A and V95A mutants occurred at between 1.5 and 3 M GdmCl with a midpoint transition of 2.09 M for V21A and 2.3 M for V95A. The {Delta}G0 value and the proportionality constant m were 7.4 kcal mol-1 and 3.5 kcal mol-1 M-1 for V21A and 5.6 kcal mol-1 and 2.5 kcal mol-1 M-1 for V95A, respectively.

The Cm of the mutated proteins V21A (2.09 M) and V95A (2.3 M) decreased significantly, compared to that of the WT protein (3.02 M). For the V95A there is also a significant decrease of the m constant.

Interactions between residues 21 and 95

The results revealed that positions 21 and 95 have critical effects on the stability of NCS. To investigate more precisely the relative role of these interactions we constructed the double mutant V21A/V95A (VVAA). The double mutant V21C/V95C (VVCC) was constructed to determine whether the hydrophobic interactions can be replaced by a disulfide bridge.

Characterization of the double mutants. For VVAA and VVCC mutants, protein on overloaded SDS–PAGE showed one well resolved band with no visible contamination. For each double mutant, mass spectrometry indicated the presence of a single species with the expected molecular weight (data not shown).

The CD spectrum of VVAA is fully superimposable with that of the WT (Figure 6AGo). Thus, the overall structure of the protein was not modified by these two substitutions.



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Fig. 6. (A) Comparison of CD spectra of the VVAA (x) and VVCC ({star}) double mutants and WT (–). (B) Baseline corrected DSC thermograms of WT (–), VVAA (x) and VVCC ({star}). (C) Thermal denaturation of the double mutant VVAA (x), VVCC ({circ}) and WT (•) monitored by tryptophan fluorescence. (D) Chemical denaturation of the double mutant VVAA (x), VVCC ({star}) and WT (•) monitored by tryptophan fluorescence.

 
Fluorescence emission spectra showed that the VVAA mutant has a maximum emission wavelength of 343 nm, which is similar to that of the WT. The solvent accessibility of this residue is clearly not affected by the two substitutions. Therefore, these two mutations did not induce any significant structural changes in the distal loop located at the bottom of the cleft. The integrity of the cleft was also checked by measuring EtBr binding. The Kd value obtained for VVAA (15 ± 1 µM) is very similar to that obtained for the WT protein.

For the VVCC mutant, mutations affect the structural features of the protein. First, the fluorescent spectral properties are modified by this substitution: the maximum wavelength is shifted from 343 nm (value obtained with the WT) to 341 nm indicating a change in the accessibility of Trp39 to solvent. Secondly, the CD spectrum could not be superimposed over the WT one (Figure 6AGo); in particular the apo-NCS specific positive contribution at approximately 223 nm is flattened. Finally, no data have been obtained concerning the integrity of the cleft, as the experimental conditions used for the Kd measurement led to protein aggregation.

Stability of mutants compared to the WT. The stability of the double mutant proteins was also evaluated by analyzing the thermal- and chemical- (GdmCl) induced denaturation measured by use of spectroscopy and calorimetry.

DSC measurements showed that double mutations VVAA and VVCC had the greatest effect on the thermal stability of the protein. The unfolding of the VVAA mutant led to a transition peak centered at 47.1°C, with a {Delta}Hcal of 85 kcal mol-1 and a {Delta}HvH / {Delta}Hcal ratio of 0.9. For the VVCC mutant the melting temperature is 36.6°C, with a {Delta}Hcal of 64 kcal mol-1 and a {Delta}HvH / {Delta}Hcal ratio of 0.64 (Table IIGo). The second scan performed after cooling showed that only 71 and 42% of the enthalpy could be recovered for VVAA and VVCC mutants, respectively.

We also monitored the thermal denaturation of the proteins by fluorescence, The Tm is 53°C for the VVAA mutant. This further supports the DSC results and confirms that the stability of NCS is strongly reduced when residues 21 and 95 are mutated. The VVCC mutant behave in an atypical manner (Figure 6CGo). As the temperature increased the maximum fluorescence emission wavelength did not change in the temperature range corresponding to the melting curve observed by DSC.

The apparent inconsistency between the results obtained by fluorescence measurement and DSC was clarified by thermodenaturation monitored by CD. The Tm (36°C) and {Delta}Hcal (60 kcal mol-1) obtained by CD measurements are fully consistent with the DSC data. This suggests that the atypical shape of the curve obtained by thermodenaturation monitored by fluorescence is due to the existence of a stable local microstructure around a tryptophan residue, probably Trp39.

Finally, we used fluorescence to monitor chemical denaturation of the double mutants (Figure 6DGo). As for single mutants, denaturation induced by GdmCl leads to a more extensive denaturation, such that the tryptophan residues are fully exposed.

For VVAA and VVCC mutants, the transitions shifted considerably towards the low denaturant concentration. The Cm values are 1.1 and 0.9 M for VVAA and VVCC mutants, respectively. The destabilization of the VVCC mutant is so large that the denaturation curve no longer shows a clear, cooperative transition. The calculated {Delta}G0 values are 3.2 and 0.63 kcal mol-1 for VVAA and VVCC mutants, respectively. Since there is no clear cooperative transition, this value represents a rough estimate of the free energy change.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
Effect of the mutations on the structure of the protein

Neither the single substitutions nor VVAA substitutions changed the overall structural features of the protein. At 25°C the fluorescence properties of these mutants are comparable to those of the WT protein, suggesting that the mutations do not significantly modify the accessibility of Trp39 to solvent, the main contributor to fluorescence. The CD spectra of the mutant proteins are identical to that of the WT protein, indicating that the mutations do not change the overall secondary structure content of the proteins. Moreover, the substitutions do not modify the integrity of the binding site as the Kd values obtained were all similar to that of the WT protein. Thus, the observed changes in protein stability are mainly related to changes in local interactions.

Effect of mutations on the thermodynamic parameters

The results obtained by calorimetry, thermal and chemical denaturation suggest that positions 67 and 97 are poorly implicated in the overall stability of NCS, as indicated by the fact that the Tm, {Delta}Hcal, Cm, {Delta}G and m values are similar to those of the WT. The small changes observed in protein stability can be directly related to the observed change in free energy as a result of the methyl substitutions (Xu et al., 1998Go).

In contrast, the V34A mutation has a significant effect on the stability of NCS, mainly due to the decrease in the m value as the Cm value for V34 is higher than for the WT. One well known cause of apparent decrease of m value is the deviation from a two-state unfolding mechanism, with the presence of one or more intermediates. While we cannot rule out that the heat denaturation process is different from the chemical denaturation process, the DSC measurements showed that the {Delta}HvH / {Delta}Hcal ratio is 1, which is characteristic of a two-state process. Lower m values may also result from a change in the exposed hydrophobic surface in the native state or in the denatured state. The fluorescence measurements indicate that, in the case of GdmCl denaturation, the tryptophans are fully exposed to the solvent in the denatured state, suggesting that there are very few, if any, residual structures for the WT. This indicates that the denatured state is equivalent in the mutant and the WT protein. The lower m value is thus probably directly related to changes in the hydrophobic surface area in the native state. Although a partially structured native protein could give such a result, this hypothesis can be ruled out because the mutants have native-like structural and functional characteristics, as attested by CD, fluorescence and EtBr binding measurements. Thus, the most probable explanation would be the existence of a native state that is more flexible than the WT one, leading to a lower m value. NMR and molecular dynamic simulations have shown that residue V34 is part of a cluster in which the dynamics of this region are highly correlated (Izadi-Pruneyre et al., 2001Go). This indicates that reducing the number of interactions in this region leads to an increase in internal motions, resulting in the destabilization of the protein. This observation supports the previously proposed hypothesis that there is a relationship between the internal flexibility and the stability of the protein (Izadi-Pruneyre et al., 2001Go).

Thermal denaturation for mutants V21A, V95A and VVAA shows a strong reduction in stability. The V21A, V95A and VVAA substitutions reduced the stability of the protein: the Tm decreases by approximately 10°C for the single mutations and by approximately 22°C for the double mutant. Previous studies on the hydrophobic effect have shown that the mean {Delta}{Delta}G values expected for a single substitution is approximately 1 kcal mol-1 (Xu et al., 1998Go). The {Delta}{Delta}G values obtained in this study are significantly higher, in particular for mutants V95A and VVAA: 4 and 6.4 kcal mol-1, respectively, suggesting a more specific effect than hydrophobic effect. Furthermore, the {Delta}HvH / {Delta}Hcal ratio is for mutant V21A decreases significantly, indicating that the transition proceeds through partly folded intermediate states. The presence of intermediate states for V21A is also revealed by the transition obtained by thermal denaturation as monitored by fluorescence which clearly showed a double transition.

The VVCC double mutant

CD measurements showed that the VVCC mutations affect the structural features of NCS. This leads to a marked destabilization of the protein. The thermogram is shifted and flattened compared to that of the WT, the midpoint temperature and the {Delta}Hcal decreases drastically. The results obtained by the chemical denaturation by GmdCl also show that the protein is drastically destabilized, as indicated by the low Cm, m and {Delta}G0 values. To control if a new disulfide bridge is formed between C21 and C95 we have analyzed by mass spectroscopy the fragments obtained after digestion by trypsin of the variant (data not shown). The mass spectrum indicated that the sample had a heterogeneous disulfide bridge content. The presence of the natural cysteine at position 93 could account for the presence of misfolded proteins induced by mismatches between cysteines. Obviously, the addition of the two cysteines at positions 21 and 95 does not allow the formation of a disulfide bridge that stabilizes NCS as do the hydrophobic interactions that they replace.

Possible other contributions to the apo-NCS stability

Although the NCS belongs to the Ig-fold family, its stability is significantly higher than the other members of this family. Although the data presented in this study point out the particular role of the V21–V95 interaction, some other interaction could also be important for the stability of the protein.

Recently, a study of the key interactions for the binding of the chromophore various mutants has been performed (Nozaki et al., 2002Go). By site-directed mutagenesis, it was clearly shown that F78 plays an important role for chromophore binding. In this study, it was proposed that the slight decrease in affinity of a mutant F78W could be related to a destabilization. However, F78 is totally exposed to the solvent, excluding any direct hydrophobic effect on protein stabilization. Moreover, MD shows that F78 undergoes a strong `flip motion’ in apo-NCS (Izadi-Pruneyre et al., 2001Go). Thus, the slight decrease in affinity for EtBr should be related to a local steric hindrance induced by the mutation F78W. This hypothesis is strengthened by the anisotropy measurements presented by Nozaki et al. (Nozaki et al., 2002Go), suggesting another position of the EtBr in this mutant. It is possible that such a steric hindrance could also induce a slight destabilization of the protein. However, this does not imply that F78 plays a particular role in the protein stabilization.

This work that reveals key hydrophobic interaction for NCS stability was performed on the apo-protein. A possible role of the natural chromophore for the stability of the holo-protein could be suggested. However, the role of the protein is to stabilize the chromophore and apo-NCS is itself a very stable protein compared to other Ig-like fold proteins. Moreover, comparison of apo-NCS and holo-NCS structures gives no indication about a possible structural change induced by ligand binding. Thus, the contribution of the chromophore to the stability of the protein should be very low. The only expected effect should come from the `Le Chatelier’ principle, due to the strong affinity of the NCS towards the chromophore.

Finally, another structure contributing to the stability and folding of Greek key structures is the Tyr-corner (Hemmingsen et al., 1994Go). Indeed, there is such an interaction in NCS on Y32. We have tested the role of this interaction on a mutant Y32F; such a mutant displayed significant decrease in stability (data not shown). This indicates that besides interaction in the common hydrophobic core, NCS displays other features common to the Ig-fold proteins.

Comparison with the core nucleus of other Ig-fold proteins

This study was initiated from data obtained by NMR and molecular dynamics (Izadi-Pruneyre et al., 2001Go) and thus, only selected residues were mutated. The results of this study should thus be compared to previous data obtained on other Ig-like proteins where identification of key residues was obtained by full site-mutation analysis. In particular, Clarke et al. (Clarke et al., 1999Go) have shown a clear correlation between domain stability and the rate of folding and, between domain stability and the population of folding intermediates. The authors propose that these results could be accounted for by the presence of a common folding nucleus. Further analysis of the {phi} values obtained from the folding kinetics measured for various mutants of the third fibronectin type III domain of human tenascin have located four positions in the central B, C, E and F strands that form a nucleus of tertiary interactions (Hamill et al., 2000Go). This nucleus has also been observed in other Ig-like proteins (Clarke et al., 1999Go; Lorch et al., 1999Go). More recently, systematic mutation of hydrophobic interactions in the core of FNfn10 gave data that support the hypothesis that interaction in the common structural core guide the folding of the Ig-like domain (Cota et al., 2001Go). Furthermore, as observed for other proteins, there is a correlation between destabilization and the presence of intermediates, the mutations V21A and V95A inducing changes in the folding process.

Although definitive proof that these residues are involved in a folding nucleus would require an analysis of the {phi} values obtained from the folding kinetics, these results suggest the existence of such a nucleus in NCS. Residues 21 and 95 occupy a central position in the cluster, they are implied in the stability of the protein and moreover, in previous studies (Izadi-Pruneyre et al., 2001Go), we have shown that these residues constitute the more rigid parts of the protein structure. This is consistent with the hypothesis proposed by Hamill et al. (Hamill et al., 2000Go) and then by Cota et al. (Cota et al., 2001Go) suggesting that the BCEF motif is an efficiently packed unit of structure and it is probably the stability that results from formation of this structure that drives the folding of Ig-like proteins. The lack of any evolutionary relationship between NCS and the other proteins strongly suggests that this hydrophobic core is characteristic of the Ig-fold itself.


    Conclusions
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusions
 References
 
We studied the structure and the stability of proteins in which these residues have been mutated and showed that they are implicated in different ways in the stability of NCS. Indeed, mutations at positions 67 and 97 have only little effect on the stability of NCS. Conversely, mutations at positions 34, 21 and 95 considerably destabilize NCS. Interestingly, these residues are located within specific structural motifs. Residue V34 is located in a structure corresponding to a Tyr-corner, a structure contributing to the stability and folding of Greek key structures (Hemmingsen et al., 1994Go). Residues V21 and V95 occupy a strategic position within the ß-sandwich where they coordinate the association between the B strand and the F strand. Stabilization between these two strands seems to be essential for the Ig-fold structure. In most other Ig-like proteins this hydrophobic core also contains a disulfide bridge, which connects the B and F strands. It has been proposed that this disulfide bridge is required to stabilize the ß-barrel. Indeed the stabilization of this type of structure does need a strong contact interaction, such an interaction being obtained either by a combination of hydrophobic interaction and a disulfide bridge as in Ig, or solely by strong hydrophobic contacts as in NCS.


    Notes
 
3 To whom correspondence should be addressed. E-mail: michel.desmadril{at}mip.u-psud.fr Back

M.Valerio-Lepiniec and M.Nicaise contributed equally to this work


    Acknowledgments
 
The authors thank Dr Charles Robert for carefully reading the manuscript. This work was supported by the Centre National de la Recherche Scientifique, a grant from the `Association pour la Recherche contre le Cancer’ and a `Programme Incitatif et Coopératif’ with the Institut Curie.


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Received May 22, 2002; revised August 7, 2002; accepted August 26, 2002.