Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany
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Abstract |
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Keywords: affinity improvement/atrazine/Fab fragment/molecular modeling/site-directed mutagenesis
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Introduction |
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The specificity of antibodies is defined by the complementarity determining regions (CDRs), which are formed by three hypervariable loops of the light chain (VL) domain (CDR L1L3) and three of the heavy-chain (VH) domain (CDR H1H3). The CDRs form exposed loops at the tip of each variable domain, and are supported by structurally conserved framework regions. Based on antibody structures resolved by X-ray diffraction and known sequences of several hundred other immunoglobulins, it was observed that the structure of most of the CDR loops is conserved within a limited repertoire and can be predicted using a small number of residues at conserved positions in both the CDRs and framework regions (Chothia and Lesk, 1987). These `canonical' structures are found in the most recently solved antibody structures, and were used to predict the antigen binding sites of different antibodies (Chothia et al., 1986
; Holm et al., 1990
; Jackson et al., 1992
; Xiang et al., 1996
; Lamminmäki et al., 1997
; Iba et al., 1998
). Since the CDR H3 shows variability not only in the amino acid sequence but also in its length, it has been difficult to establish relationships between sequence and tertiary structure. Recently, empirical rules to predict the CDR H3 tertiary structure have been established (Shirai et al., 1996
, 1999
). These `H3 rules' classify the backbone conformations of the bases of CDR H3 into two forms, kinked and extended, depending on the nature of the residues at the positions H94 and H101. Analysis of antibodies of known three-dimensional structures showed that the length of CDRs L1 and H2 correlate with the type of antigen recognized (Vargas-Madrazo et al., 1995
). Antibodies with long loops in CDRs L1 and H2 are preferentially specific for small molecules (haptens), which are bound in either groove or cavity-type antigen-binding sites. In contrast, antibodies with short loops in CDRs L1 and H2 preferentially bind to surfaces of large antigens in flat or slightly concave binding sites.
We took advantage of such information for a rational design of the anti-atrazine antibody Fab fragment K411B to investigate the molecular basis of antibody specificity and to improve its affinity by site-directed mutagenesis. Atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) and its many analogs constitute the triazine family of herbicides, which are selective inhibitors of photosynthesis in weed species. Because of its persistence and widespread application, triazine causes problems as a contaminant in surface and ground waters (Schlaeppi, et al., 1989; Giersch and Hock, 1990
; Goodrow et al., 1990
). The Fab fragment K411B was derived from the monoclonal antibody K4E7 (isotype: IgG2b with
light chain), which was generated by immunization with 4-chloro-6-(ethylamino)-1,3,5-triazine-2-(6-aminohexanecarboxylic acid) conjugated to keyhole limpet hemocyanin (Giersch, 1993
; Kramer and Hock, 1996
). Although the immuno conjugate carries a free chlorine and an ethyl group, the highest affinities of K4E7 were observed towards s-triazines, which have at least one isopropyl group (Giersch, 1993
).
In the present study, a three-dimensional model of the variable fragment (Fv) of K411B was constructed based on the homology to immunoglobulins of known structures and the canonical structure classes for the CDRs (Chothia and Lesk, 1987; Chothia et al., 1989
, Al-Lazikani et al., 1998). Molecular mechanics and dynamics were used to probe and refine the position of the manually-docked hapten molecule (Chothia et al., 1986
; Lim and Herron, 1995; Lämminmäki et al., 1997
). Cross-reactivity data of different triazines were used for the construction and evaluation of the structure model of the complex. Mutations of both heavy- and light-chain amino acids in the predicted binding pocket were modeled and the corresponding effect was experimentally verified by capture ELISA using two haptens, 4-chloro-6-(isopropylamino)-1,3,5-triazine-2-(6-aminohexanecarboxylic acid) (iPr/Cl/C6) and 4-amino-6-chloro-1,3,5-triazine-2-(6-aminohexanecarboxylic acid) (H/Cl/C6), and by surface plasmon resonance (SPR). The predicted structure model could help developing strategies to improve the sensitivity of a direct competitive ELISA for the determination of atrazine.
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Material and methods |
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Rabbit anti-mouse IgG (RAM) and horseradish peroxidase (HRP) were obtained from Sigma (Buchs, Switzerland). s-Triazines (Figure 1A) were purchased from Fluka (Deisenhofen, Germany). The synthesis of s-triazines with n-propyl, n-butyl, sec-butyl or tert-butyl group and the carboxylated s-triazine derivatives was performed as described elsewhere (Goodrow et al., 1990
; Weller and Niessner, 1997
). The HRPhapten and BSAhapten conjugates were prepared according to the method described by Wittmann (Wittmann and Hock, 1989
). The solutions of the HRPhapten conjugate were normalized by determining the enzyme activity and the absorption of HRP at 403 nm.
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Escherichia coli strain DH5 was used as the cloning host and E.coli JM 101 as expression host for soluble Fab fragments. The cultivation medium was Luria broth (LB) supplemented with 100 µg/ml ampicillin. The vector pK411B was obtained by cloning the cDNAs encoding the wild-type heavy and light chains into the vector pASK85 under the control of a tet-promotor. The VH gene was inserted between the restriction sites PstI and BstEII in pASK85, and the VL gene was introduced between the restrictions sites SacI and XhoI, respectively.
Site-directed mutagenesis and sequencing
Site-directed mutagenesis was carried out by means of the QuikChange Site-directed Mutagenesis Kit (Stratagene, La Jolla, CA) according to the protocols with primers containing the desired mutations. The cDNA of the mutants were isolated with the Spin Miniprep DNA Isolation Kit (Qiagen, Hilden, Germany) according to the user's manual and checked with agarose gel-electrophoresis. Sequencing reactions were performed with the Big Dye Sequencing Kit with AmpliTaq-DNA polymerase according to the manufacturer's instructions (Applied Biosystems, Frankfurt, Germany).
Expression and purification
The expression vectors were transformed into the E.coli strain JM101. Overnight cultures were diluted 1:50, grown at 25°C in a shaking flask to an OD550 of 0.81.0, after which anhydrotetracycline (IBA, Göttingen, Germany) was added to a final concentration of 0.2 µg/ml and growth was allowed to continue for 4 h. The cells were harvested by centrifugation (5000 g, 15 min, 4°C) and resuspended in 50 mM PBS containing 300 mM NaCl. After addition of lysozyme to a final concentration of 0.5 mg/ml and incubation at room temperature for 20 min, the suspension was sonicated carefully to isolate the Fab fragments which were expressed in the periplasmic space of E.coli.
Purification of the Fab fragments was performed by IMAC on TALONTM chromatography matrix (Clontech, Heidelberg, Germany). Four milliliters of the matrix was loaded into a PD-10 column (Pharmacia, Freiburg, Germany) and equilibrated with 50 mM PBS containing 300 mM NaCl. Ten milliliters of periplasmic extract from a 1 l culture was applied to the column. After washing with 40 ml of the same buffer the Fab fragments were eluted from the column with 100 mM imidazole in 50 mM PBS and 300 mM NaCl. Removal of the imidazole was performed by gel filtration on Sephadex G-25 in a PD-10 column (Pharmacia, Freiburg, Germany).
ELISA
The IC50 values (the concentration of the hapten required to reduced the signal by 50%) were determined by a competitive ELISA. In all steps a volume of 200 µl/well was employed. After each incubation the microtiter plate (MaxiSorp; NUNC, Roskilde, Denmark) was washed six times with PBS pH 7.2 supplemented with 0.1% Tween 20. The microtiter plate wells were first coated overnight at 4°C with rabbit anti antibody (RAM; 1µg/ml in 50 mM carbonate buffer pH 9.6). The Fab fragments were then bound to the RAM by incubation for 2 h at room temperature in PBS buffer. The tracer was mixed with a dilution series of the hapten and incubated in the wells at room temperature for 1 h. After incubation of 200 µl of the color reagent tetramethylbenzidine and hydrogen peroxide in acetate buffer pH 5.5 for 15 min and stopping the reaction with 100 µl of 1 M H2SO4, the absorbance in each well was read at 450 nm. Cross-reactivity was calculated as the ratio of the IC50 value for atrazine to the IC50 value of the cross-reacting s-triazines.
The relative affinity of each mutant compared to the wild-type was determined by capture ELISA using the hapten iPr/Cl/C6 and H/Cl/C6 (Figure 1B and C). Microtiter plate wells were first coated with 0.4 µg/l RAM. In each well the cell extract containing Fab fragments of a clone, 5-fold diluted in 50 mM carbonate buffer of pH 9.6, was then incubated for 2 h. Each haptenHRP tracer was diluted in PBS buffer containing 0.5% casein and incubated in the wells at room temperature for 1 h. The color reaction was performed as described above. The absorbance of each mutant at 450 nm was linearly scaled to that of the wild-type Fab fragment to obtain the relative affinity.
Kinetic measurements by SPR
Dissociation and association constants of the purified Fab fragments were measured with a Biacore 3000 instrument (Biacore AB, Freiburg, Germany) (Johnsson et al., 1991; O'Shannessy et al., 1992
). The iPr/Cl/C6BSA conjugate or BSA as control was immobilized on the sensor chip CM5 (Biacore AB) by the amine coupling method. Briefly, 50 µl of a 1:1 mixture of 0.1 M N-hydroxysuccinimide (NHS) and 0.2 M N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) was pumped across the chip to activate the carboxymethylated dextrane surface. Subsequently, the iPr/Cl/C6BSA conjugate or BSA in 10 mM sodium acetate (pH 4.5) was pumped across the activated surface to give a resonance response of 670 RU for the iPr/Cl/C6BSA conjugate in the first cell and 620 RU for BSA in the second cell. The residual NHS esters were inactivated with ethanolamine. The purified Fab fragments at different concentrations (50, 100, 160, 200, 300 and 400 nM) were injected via the sample loop of the system. Measurements of SPR were performed at a constant flow rate of 30 µl/min in HBS, which consisted of 10 mM HEPES (pH 7.4), 150 mM NaCl and 0.05% Tween 20, at 25°C. The regeneration of the surface was done by injection of 30 µl of 100 mM HCl. Data were evaluated using the BIAevaluation 3.0 program (Biacore AB).
Structure modeling
The variable domain of the anti-atrazine antibody fragment K411B was constructed based on the crystal structures of the VH domain of an anti-dansyl antibody (PDB entry: 1dlf; Nakasako et al., 1999) and the VL domain of the Fab fragment 50.1, an antibody specific for a 16-residues peptide of HIV gp120 (PDB entry: 1ggi; Rini et al., 1993
). The sequence identity between the anti-dansyl antibody 1dlf and K411B, excluding CDR H3, is 86%. The main chain conformation of CDR H1 is defined by its length and by amino acids at the positions H26, H27, H29, H34 and H94 (Chothia and Lesk, 1987
). The amino acids of K411B at these positions are identical to those in 1dlf, with the exception of position 94, which is Arg in K411B, but Gly in 1dlf (Figure 2
). However, CDR H1 of 1dlf belongs to canonical class 1, which permits Arg or Gly at position H94. As a result, we predicted that the main chain conformation of CDR H1 of K411B would be similar to that of 1dlf. The conformation of CDR H2 is determined by its length and residues at the positions H55 and H71. CDR H2 of 1dlf and K411B have the same length and residues at both positions, and their sequences differ only in the position H57 (Figure 2
). CDR H2 of K411B and of 1dlf were, therefore, predicted to share the same main chain conformation (canonical class 4).
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The VL domain of K411B shares an overall 86% sequence identity with 1ggi. The residues at the positions L2, L25, L27b, L33 and L71 determine the conformation of 15-residues long V CDR L1, which is assigned to canonical class 5 (Martin and Thornton, 1996
; Al-Lazikani et al., 1997
). CDR L1 of K411B fulfils completely the criteria for this canonical structure, and therefore, was predicted to adopt the same main chain conformation as that of 1ggi. CDRs L2 (class 1) and L3 (class 1) also match a known canonical class for each of these CDRs (Chothia and Lesk, 1987
). After constructing the backbone of the Fv fragment, the side chains were replaced using rotamer libraries and algorithm implemented in the Homology package of InsightII 97.0 (MSI, San Diego, CA).
Docking of hapten
Our ELISA measurements (see below, Table I) showed that K411B exhibited a 6-fold higher affinity towards the derivative iPr/Cl/C6 than towards atrazine. Therefore, in our docking studies, we used iPr/Cl/C6 as hapten to identify the recognition site of the long alkyl spacer of the hapten. The structure of iPr/Cl/C6 was constructed using the module Builder in InsightII 97.0 (MSI). The partial charges of the hapten were assigned and the structure was optimized using the module AMPAC3/AM1 of InsightII 97.0. The hapten was docked manually into the binding site of K411B. Using interactive graphics and the hard-sphere approximation for atoms, we found that the hapten could be fitted into the binding site such that the isopropyl residue of the hapten is buried deep in the binding site, where it is surrounded by hydrophobic protein residues. The end of the C6-spacer was left accessible to the solvent, consistent with the fact that the hapten was coupled to its carrier protein via the carboxyl group of the spacer during immunization. Since the rotational freedom of the hapten is restricted by the protein environment, three plausible initial complexes were examined, corresponding to different orientations of the chlorine atom of the hapten. The haptenantibody complexes were relaxed by energy minimization and subsequent molecular dynamic simulations in vacuo using the CVFF force field (Dauber-Osguthorpe et al., 1988
). During the initial equilibration phase, the complexes were heated in three intervals of 2 ps each to 10, 100 and 200 K, and 10 ps at 298 K, followed by a 10 ps production run at 298 K, at a step size of 1 fs. The backbone atoms of the CDR loops (residues L24L34, L50L56, L89L97, H26H35, H50H58) were constrained with a force constant of 10 kcal mol1 Å2. Due to uncertainty and the higher structure variability of CDR H3, no constraints were applied to the residues H94H102 of this CDR. Non-bonded interactions were calculated using the cell multipole method (Greengard and Rokhlin, 1987
; Schmidt and Lee, 1991
; Ding et al., 1992
). Molecular dynamics simulations were performed using the NVT ensemble of the Discover 3.0.0 package (MSI).
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Results |
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The K411B Fab fragment expression unit in pASK85 expression vector was transformed into the E.coli JM 101 strain. The Fab fragment was secreted as active and soluble protein into the periplasmic space after induction with anhydrotetracycline. It was purified in two steps by means of the IMAC on TALONTM matrix followed by gel filtration on Sephadex G-25. The production levels obtained were 34 mg purified Fab from a 1 l E.coli culture.
Cross-reactivity data
The binding of the antibody Fab fragment K411B to several s-triazines was measured by competitive ELISA. The structural differences among the s-triazines and the cross-reactivity data are listed in Table I. The cross-reactivity patterns of K411B are similar to those of the monoclonal antibody K4E7 (Giersch, 1993
).
In the interpretation of the cross-reactivity data, it should be noted that due to the symmetry of the triazine ring, positions R1, R2 and R3 are equivalent (Figure 1A). We assume that the substituents at R1, R2 and R3 will bind to three different pockets in the binding site of K411B. All derivatives with alkylamino groups other than isopropylamino at R1 and R3 show lower cross-reactivity compared to atrazine (iPr/Cl/Et). This indicates that there is a binding pocket highly specific to the isopropylamino group. Replacing the isopropylamino group at R1 by smaller ethylamino group or amino group while keeping the same alkylamino group at R3 leads to a decrease in cross-reactivity (iPr/Cl/C6 > Et/Cl/C6 > H/Cl/C6). Bulkier alkyl residues than isopropyl at R1 are bound with 6- to 8-fold lower cross-reactivity (iPr/Cl/tBu > sBu/Cl/tBu > tBu/Cl/tBu).
Triazines with isopropyl residue at R1 and alkyl residues bulkier than ethyl at R3 are bound with higher affinity: the Fab fragment K411B showed 2-fold higher cross-reactivity to the haptens iPr/Cl/iPr, iPr/Cl/sBu and iPr/Cl/tBu compared to iPr/Cl/Et. This suggests that the binding pocket around R3 is not specific to the ethyl group, but is large enough to accommodate bulkier alkyl residues. The antibody shows even higher affinity for derivatives with alkyl residues longer than ethyl at R3. The affinity for these derivatives increases concomitantly with the increase in length of the alkyl residue at R3 (iPr/Cl/C6 > iPr/Cl/nBu > iPr/Cl/nPr > iPr/Cl/Et). The binding site of K411B is very chlorine specific, since substitution of chlorine at the position R2 either by hydroxyl, methoxy or thiomethyl groups leads to a two orders of magnitude lower cross-reactivity.
Construction of the structure model
To investigate the molecular basis of the observed specificity, a three-dimensional model of the antigen-binding site of K411B was constructed employing the combination of two techniques shown previously to give reliable results: the identification of canonical structures (Chothia and Lesk, 1987; Chothia et al., 1989
) and molecular dynamics simulation (Chothia et al., 1986
; Lamminmäki et al., 1997
).
From three different initial orientations of the chlorine atom of iPr/Cl/C6, only one model was obtained in which the hapten was still bound deep in the cleft. In the other two complexes, the hapten drifted out of the cleft during the simulation. The RMSD of the backbone atoms of CDRs L1L3, H1 and H2 between the starting and final conformation of the selected model after energy minimization and MD simulations was 1.4 Å. The most significant structural changes during the simulation occurred in CDR H3 with RMSD of the backbone atoms of 3.2 Å between the starting and final conformation, while the KG kinked form of the C-terminal conformation of CDR H3 (Shirai et al., 1999) was retained. The pseudo-dihedral angle formed by the four successive C
atoms at the position C-1, C-2, C-3 and C-4 (C is the position H102 of the CDR H3), denoted
stem, changed from 37.2° to 24.8° after MD simulation. The
and
angles of GlyH100a changed from 6.2° and 173.1° to 25.7° and 155.3°, respectively.
stem, as well as
and
angles for GlyH100a of K411B were in the range of the values observed in known CDR H3 conformations with the KG kinked base structure (Shirai et al., 1999
).
The binding site of K411B
Five CDRs, L1, L3, H1, H2 and H3, line a deep cleft between the heavy- and light-chain variable domains (Figure 3). The cleft is ~12 Å deep and ~10 Å wide, and constitutes the putative hapten-binding site of K411B. The triazine ring is sandwiched between TyrL96, HisH95 and GlyH100a (Figure 4
). TyrL96 has its ring plane oriented almost parallel to the triazine ring. According to the model of the complex, three hydrogen bonds are formed upon binding of the hapten to the antibody. The hydroxyl group of SerL91 and the carbonyl oxygen of GlyH100a make hydrogen bonds with the amino group of the C6-spacer of iPr/Cl/C6, whereas the carboxyl oxygen of GlnL89 seems to be hydrogen-bonded to the amine of the isopropylamino group of the hapten.
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Modeling of mutants and experimental verification
Site-directed mutagenesis was performed in three regions of the binding pocket of K411B: in the binding pocket of the chlorine atom, of the spacer and of the isopropylamino group of the hapten. Amino acid substitutions were carried out for residues in the CDRs as well as in the framework regions. The relative affinity of the mutants compared to the wild-type Fab fragment was determined by capture ELISA (Burks et al., 1997; Weller and Niessner, 1997
) using the haptens iPr/Cl/C6 and H/Cl/C6. A low or absent ELISA signal means that the off-rate is too fast for the Fab mutants to remain bound to a significant extent during the incubations and washing steps, but does not necessarily imply a complete loss of binding (Burks et al., 1997
).
The residues which form the binding pocket of the chlorine atom of the hapten are TrpH33, GluH50, HisH95 and TyrL96. Altering the amino acids in these positions resulted in reduced relative affinity (Table II). Substitution of GluH50 with any other amino acid diminished the binding of the haptens iPr/Cl/C6 and H/Cl/C6 to the antibody. Only one mutation, GluH50Gln, retained the binding of the hapten, albeit with a significant decrease in relative affinity. By removing the putative hydrogen bond between TyrL96 and GluH50, and by replacing TyrL96 with phenylalanine, the relative affinity was reduced 3-fold. Exchanging TrpH33 by amino acids with a smaller side chain (Tyr, Phe or Leu) reduced the relative affinity significantly. However, replacement of HisH95 by phenylalanine had only a slight effect on the affinity, whereas the substitution of HisH95 by tyrosine resulted in a mutant, which bound the hapten with 2-fold less relative affinity.
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According to the model, residue GlnL89 is in direct contact with the isopropylamino group of iPr/Cl/C6. The side chain of GlnL89 contributes to lining the hydrophobic wall making van der Waals contacts with the isopropyl group, while its carboxyl oxygen might form a hydrogen bond with the amino group. In an attempt to increase the binding of the hapten, GlnL89 was replaced by glutamic acid. Compared to the wild type, this mutation increased the relative affinity to the hapten iPr/Cl/C6 slightly, whereas it showed a 2-fold higher relative affinity towards H/Cl/C6 as evidenced by capture ELISA.
An additional single amino acid substitution was introduced into the mutant GlnL89Glu at position H37 by replacing the highly conserved valine by isoleucine. ValH37 lines the bottom of the binding pocket and makes van der Waals contacts with one methyl group of the isopropyl group of iPr/Cl/C6. The substitution led to a double mutant, GlnL89Glu/ValH37Ile, which exhibited a higher relative affinity towards iPr/Cl/C6 than the single mutant GlnL89Glu (Table II). The relative affinity of the mutant GlnL89Glu/ValH37Ile towards H/Cl/C6 was even increased 4-fold compared to the wild-type. However, the single mutant ValH37Ile showed only a slight effect on relative affinity towards both haptens. In an attempt to alter the backbone conformation of CDR L3, which might lead to higher affinity due to improved contacts of the backbone or the side chains of residues of CDR L3 with the hapten, mutations were performed simultaneously for residues in positions L3 and L4 of the mutant GlnL89Glu/ValH37Ile, which are in direct contact with CDR L3. The residue GluL3 was replaced by Glu, Gln, Val or Leu, and LeuL4 by Leu or Met. The chosen residues are often found in the respective positions. Only the triple mutant GlnL89Glu/ValH37Ile/GluL3Val exhibited increased relative affinity towards iPr/Cl/C6 compared to GlnL89Glu/ValH37Ile.
Analysis of the kinetics of binding
In order to verify the observed increase in relative affinity, the binding kinetics of the wild-type Fab fragment K411B and the mutants were determined by SPR using the Biacore 3000. The iPr/Cl/C6BSA conjugate was immobilized on the sensor surface and the Fab fragments were passed continuously over the conjugate. Following Biacore analysis of a range of concentrations, association and dissociation rates were calculated using the 1:1 Langmuir binding model. Whereas the ELISA results indicate only 1.11.3-fold improvements in the relative affinity for iPr/Cl/C6, the results obtained by Biacore analysis indicate 25-fold increase in absolute affinity for iPr/Cl/C6 (Table III). Since the result obtained by capture ELISA might not be strictly quantitative, the values from Biacore analysis should be considered to be more accurate. Combination of mutations with 1.5- to 2-fold higher affinity, ValH37Ile and GlnL89Glu showed an additive effect on affinity resulted in 4-fold higher affinity of the mutant GlnL89Glu/ValH37Ile.
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Discussion |
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Based on the model of the complex, residues which may contribute to the binding of the hapten iPr/Cl/C6 were identified. To test this prediction, some of these residues were exchanged by site-directed mutagenesis either to eliminate hydrogen bonds or to alter van der Waals interactions with the hapten.
Replacing residues in direct contact with the chlorine atom of the hapten resulted in a dramatic decrease or loss of binding affinity. A significant reduction in the relative affinity was observed when GluH50 was replaced by glutamine. Glutamine might, in principle, be capable of preserving all steric interactions. However, the reduced affinity might be a direct consequence of the removal of the electrostatic interaction between GluH50 and ArgH52. This interaction seems to be required for the correct positioning of GluH50, thus exposing only the hydrophobic Cß and C atoms of its side chain to contact with the chlorine atom of the hapten. In addition, the uncharged glutamine may be unable to position TyrL96 correctly. This would happen if the amide nitrogen and not the carbonyl oxygen of GlnH50 is positioned closest to the phenolic OH group of TyrL96, or if the higher strength of a charged hydrogen bond is required. To verify the latter prediction, phenylalanine was introduced in position L96, which resulted in a 3-fold decrease in relative affinity. Our data also show that the replacement of TrpH33 by amino acids with less bulkier side chains (Tyr, Phe and Leu) led to a significant decrease in the relative affinity, which is most likely due to the loss of van der Waals contacts between these residues and the hapten.
The recognition of the alkyl-spacer of iPr/Cl/C6 by the binding site of K411B was mainly attributed to the interaction with the side chain of PheL32. Replacing PheL32 by leucine reduced the van der Waals contacts with the spacer of the hapten, and therefore decreased the relative affinity. The weaker binding may also be due to a conformational change in the CDR H3, since PheL32 is in direct contact with the backbone of the CDR H3.
Substitution of Gly by any other amino acid would substantially change the conformation of the CDR H3, since GlyH100a defines the KG kinked structure of the C-terminal region of the CDR H3 as suggested by Shirai et al. (Shirai et al., 1999). Alternatively, the dramatic decrease in affinity by the mutation GlyH100aAla or the loss of binding by the mutation GlyH100aSer may be due to steric conflicts with the hapten introduced by the larger size of the side chain of Ala or Ser, which would be oriented towards the binding site.
The increase in affinity of an antibodyantigen interaction by site-directed mutagenesis has been previously reported for hapten-specific antibodies (Riechmann et al., 1992; Ruff-Jamison and Gleney, 1993
). In their study, a 3- to 10-fold increase in affinity could be achieved by replacing side chains in contact or even non-contact regions of the hapten-binding site. A mutant antibody with higher affinity could be obtained in our study as well, as determined by capture ELISA (Table II
). As shown by Biacore analysis, the mutation GlnL89Glu resulted in a 2-fold increase in the affinity of K411B toward iPr/Cl/C6. By replacing the neutral residue GlnL89 by the negatively-charged glutamic acid, an increase in binding due to electrostatic interactions with the amine of the isopropylamino group of iPr/Cl/C6 could be expected. However, both alkylamino substituents of iPr/Cl/C6 are only slightly basic and nucleophilic due to mesomeric stabilization of the free electron pairs, so that a formation of a salt bridge is not likely to occur. Since Glu and Gln may occupy similar positions and provide similar hapten contacts as suggested by modeling experiments (data not shown), we must consider the possibility of a bidentate binding of both carboxylate oxygens of GluL89 to the amine of the isopropylamino group, which may be responsible for the increase in affinity.
A 4-fold increase in affinity compared to the wild-type was achieved by introducing an additional mutation ValH37Ile into the mutant GlnL89Glu (Table III). A likely explanation for the increased affinity is better contact with the bigger residue isoleucine or the arrangement of residues TrpH47, PheH100b and PheL98 in the surrounding of ValH37 upon replacing of valine by isoleucine that may provide better contacts with the hapten iPr/Cl/C6. An additional mutation GlnL3Val further increased the affinity towards iPr/Cl/C6. The triple mutant GlnL89Glu/ValH37Ile/GlnL3Val showed 5-fold better affinity than the wild-type. Changes in affinity due to mutations within the N-terminal regions of the heavy or light chains have been noticed also in other studies (Short et al., 1995
; Hemminki et al., 1998
).
We also investigated the influence of the increase in affinity on the sensitivity of a competitive ELISA for atrazine using iPr/Cl/C6HRP as tracer. Since the higher affinity was mainly due to better recognition of the isopropyl residue of the hapten, it could be suggested that the relative affinity between unlabeled hapten (atrazine) and labeled hapten (iPr/Cl/C6HRP) was not changed. Therefore, no improvement in the sensitivity of the competitive ELISA was obtained using the mutant GlnL89Glu/ValH37Ile/GlnL3Val (data not shown).
In summary, molecular modeling combined with site-directed mutagenesis allowed us to identify residues responsible for the binding of atrazine by the Fab fragment K411B. In addition, we have been able to engineer a Fab fragment that is capable of recognizing the hapten better than the wild-type Fab fragment. Based on the model, residues responsible for the spacer recognition were identified. Reduced spacer recognition of the hapten iPr/Cl/C6, which is used to synthesize the tracer in ELISA for atrazine, was achieved by the mutation PheL32Leu. This offers the possibility of increasing the sensitivity of a direct competitive ELISA for the determination of atrazine due to the decrease in the relative affinity to the tracer with respect to the relative affinity to atrazine.
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Notes |
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Acknowledgments |
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References |
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Bernstein,F.C., Koeztle,T.F., Williams,G.J.B., Meyer,E.F.Jr, Brice,M.D., Rodgers,J.R., Kennard,O., Shimanouchi,T. and Tasumi,M. (1977) J. Mol. Biol., 112, 525542.
Burks,E.A., Chen,G., Georgiou,G. and Iverson,B.L. (1997) Proc. Natl Acad. Sci. USA, 94, 412417.
Chothia,C. and Lesk,A.M. (1987) J. Mol. Biol., 196, 901917.[ISI][Medline]
Chothia,C., Lesk,A.M., Levitt,M., Amit,A.G., Mariuzza,R.A. Philips,S.E.V. and Poljak,R.J. (1986) Science, 233, 755.[ISI][Medline]
Chothia,C., Lesk,A.M., Tramontano,A., Levitt,M., Smith-Gill,S.J., Air,G., Sheriff,S., Padlan,E.A., Davies,D., Tulip,W.R., Colman,P.M., Spinelli,S., Alzari,P.M. and Poljak,R.J. (1989) Nature, 342, 877883.[CrossRef][ISI][Medline]
Dauber-Osguthorpe,P., Roberts,V.A., Osguthorpe,D.J., Wolff,J., Genest,M. and Haglar,A.T. (1988) Proteins, 4, 3147.[ISI][Medline]
Ding,H.Q., Karasawa,N. and Goddard,W.A.,III (1992) J. Chem. Phys., 97, 43094315.[CrossRef][ISI]
Dougan,D.A., Malby,R.L., Gruen,L.C., Kortt,A.A. and Hudson,P.J. (1998) Protein Eng., 11, 6574.[Abstract]
Giersch,T. (1993) J. Agric. Food Chem., 41, 10061011.[ISI]
Giersch,T. and Hock,B. (1990) Food Agric. Immunol., 2, 8597.
Goodrow,M.H., Harrison,R.O. and Hammock,B.D. (1990) J. Agric. Food Chem., 38, 990996.[ISI]
Greengard,L. and Rokhlin,V.I. (1987) J. Comp. Phys., 73, 325348.[ISI]
Hemminki,A., Niemi,S., Hoffrén, A.-M., Hakalahti,L., Söderland,H. and Takkinen,K. (1998) Protein Eng., 11, 311319.[Abstract]
Herron,J.N., He,X.M., Mason,M.L. Voss,E.W.,Jr and Edmunson,A.B. (1989) Proteins, 5, 271280.[ISI][Medline]
Holm,L., Laaksonen,L., Kaartinen,M., Teeri,T.T. and Knowles,J.K.C. (1990) Protein Eng., 3, 403409.[Abstract]
Iba,Y., Hayashi,N., Sawada,I., Titani,K. and Kurosawa,Y. (1998) Protein Eng., 11, 361370.[Abstract]
Jackson,T., Morris,B.A., Martin,A.C.R., Lewis,D.F.V. and Sanders,P.G. (1992) Protein Eng., 5, 343350.[Abstract]
Johnsson,U., Fagerstam,L., Ivarsson,B., Johnsson,B., Karlsson,R., Landh,K., Lofas,S., Persson,B., Roos,H. and Ronnberg,I. (1991) Biotechniques, 11, 620627.[ISI][Medline]
Kabat,E.A., Wu,T.T., Perry,H.M., Gottesman,K.S. and Foeller,C. (1991) Sequences of Proteins of Immunological Interest, 5th edn. NIH Publication No. 91-3242, US Department of Health and Human Services, Washington, DC.
Kramer,K. and Hock,B. (1996) Food Agric. Immunol., 8, 97109.[ISI]
Lamminmäki,U., Villoutreix,B.O., Jauria,P., Saviranta,P. Vihinen,M., Nilsson,L., Teleman,O. and Lövgren,T. (1997) Mol. Immunol., 34, 12151226.[CrossRef][ISI][Medline]
Lim,K. and Heron,J.N. (1995) Biochemistry, 34, 69626974.[ISI][Medline]
Martin,A.C.R. and Thornton,J.M. (1996) J. Mol. Biol., 263, 800815.[CrossRef][ISI][Medline]
Nakasako,M., Takahashi,H., Shimba,N., Shimada,I. and Arata,Y. (1999) J. Mol. Biol., 291, 117134.[CrossRef][ISI][Medline]
Novotny,J. (1991) Mol. Immunol., 28, 201207.[CrossRef][ISI][Medline]
O'Shannessy,D.J., Brigham-Burke,M. and Peck,K. (1992) Anal. Biochem., 205, 132136.[ISI][Medline]
Rees,A.R., Staunton,D., Webster,D.M., Searle,S.J., Henry,A.H. and Pederson,J.T. (1994) Trends Biotechnol., 12, 199207.[ISI][Medline]
Riechmann,L., Weill,M. and Cavanagh,J. (1992) J. Mol. Biol., 224, 913918.[ISI][Medline]
Rini,J.M., Stanfield,R.L., Stura,E.A., Salinas,P.A. Profy,A.T. and Wilson,I.A. (1993) Proc. Natl Acad. Sci. USA, 90, 93256329.[Abstract]
Ruff-Jamison,S. and Glenney,J.R.,Jr (1993) Protein Eng., 6, 661668.[Abstract]
Schlaeppi,J.M., Föry,W. and Ramsteiner,K. (1989) J. Agric. Food Chem., 37, 15321538.[ISI]
Schmidt,K.E. and Lee,M.A. (1991) J. Stat. Phys., 63, 12231235.[ISI]
Shirai,H., Kidera,A. and Nakamura,H. (1996) FEBS Lett., 399, 18.[CrossRef][ISI][Medline]
Shirai,H., Kidera,A. and Nakamura,H. (1999) FEBS Lett., 455, 188197.[CrossRef][ISI][Medline]
Short,M.K., Jeffrey,P.D., Kwong,R.F. and Margolies,M.N. (1995) J. Biol. Chem., 270, 2854128550.
Trinh,C.H., Hemmington,S.D., Verhoeyen,M.E. and Phillips,S.E.V. (1997) Structure, 5, 937948.[ISI][Medline]
Vargas-Madrazo,E., Lara-Ochoa,F. and Almagro,J.C. (1995) J. Mol. Biol., 254, 497504.[CrossRef][ISI][Medline]
Weller,M.G. and Niessner,R. (1997) SPIE, 3105, 341352.
Wittmann,C. and Hock,B. (1989) Food Agric. Immunol., 1, 211224.
Xiang,J., Sha,Y., Prasad,L. and Delbaere,L.T.J. (1996) Protein Eng., 9, 539543.[Abstract]
Received June 8, 2001; revised December 5, 2001; accepted December 10, 2001.