Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060-0812, Japan and 1 Department of Chemistry, University of Florida, Gainesville, FL 32611, USA
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Abstract |
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Keywords: antibodies/fluorescence/mutagenesis/pyrimidine (64) pyrimidone photoproduct/surface plasmon resonance
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Introduction |
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A variety of techniques have been used to examine the details of proteinDNA interactions in the series of anti-(64) photoproduct antibodies. Computer modeling predicted that the Fv structures of antibodies 64M2, 64M3 and 64M5 would be highly similar and that interactions with the (64) photoproduct itself would involve mainly non-charged interactions since the center of the combining site was predicted to possess few ionizable amino acid side chains (Morioka et al., 1998). On the other hand, it was observed experimentally that the affinity of the 64M5 antibody for oligonucleotides containing a central (64) photoproduct increased with increasing lengths up to a hexanucleotide (Kobayashi et al., 1998a
). This suggests that electrostatic contacts might be formed between antibody side chains and phosphate groups on flanking regions of the oligonucleotides and these have been detected experimentally by 31P NMR (Torizawa et al., 1998
). A strongly cationic patch was observed on the 64M5 VH surface approximately 20 Å from the center of the combining site. Single and multiple alanine replacements for the four lysine residues making up this cationic patch were created and the properties of these mutants suggested that the region functioned in the wild-type protein as an `electrostatic steering' element during the association phase of DNA binding, although these amino acid side chains apparently did not interact directly with the antigen after binding had occurred (Kobayashi et al., 1998b
). Mutagenesis studies of CDR loop amino acids that differed between the 64M5 and 64M2 antibodies suggested that these proteins undergo conformational changes upon antigen binding and that the greater propensity to undergo these conformational changes is the major reason for the higher affinity of the 64M5 antibody (Kobayashi et al., 1999
). The X-ray crystal structures of the free 64M5 Fab fragment and the 64M2 Fab fragment complexed with a d(TT) (64) photoproduct dimer have been solved recently (Yokoyama,H., Mizutami,R., Satow,Y., Komatsu,Y., Ohtsuka,E. and Nikaido,O., manuscript in preparation). These experimental structures were very similar to those predicted by computer modeling and comparison of the bound and free states showed the conformational changes expected from earlier studies. The apparent stacking interaction between the Trp H33 side chain and the 3' pyrimidone nucleotide was a particularly striking feature of the 64M2 complex structure (Figure 1
). This Trp residue, located in VH CDR1, is conserved in the 64M2, 64M3 and 64M5 antibodies and also in a number of other murine anti-DNA antibodies of the
2a and
2b subclasses (Kabat et al., 1992
).
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In this paper, we examine the role of Trp H33 in photoproduct binding by the 64M5 scFv. Mutants were constructed in which this residue was substituted with phenylalanine, tyrosine or alanine. While the W-H33-F and W-H33-Y mutants retained a large fraction of the wild-type binding affinity for DNA's containing a (64) photoproduct, the W-H33-A substitution dramatically diminished antigen binding. Pyrimidine (64) pyrimidone photoproducts are fluorescent, and this property was exploited to probe the local environment of the bound antigen in the wild-type and the mutant scFvs. Taken together, our results indicate that Trp H33 plays a key role in DNA photoproduct binding by the 64M5 antibody, most likely by -stacking interactions.
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Materials and methods |
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Restriction and DNA modifying enzymes were purchased from Takara Shuzo, New England Biolabs, Bethesda Research Labs, Stratagene, Toyobo or Boehringer Mannheim. Reagents were obtained from Wako Pure Chemical Industries, Nacalai Tesque or Sigma Chemical and were used as received. DNA phosphoramidite reagents were obtained from Perkin Elmer Applied Biosystems. Routine cloning was performed according to Sambrook et al. (1989).
Preparation of oligonucleotides
Solid-phase oligonucleotide synthesis was carried out on an Applied Biosystems Model 394 DNA/RNA synthesizer using standard ß-cyanoethyl chemistry according to the manufacturer's protocol. Oligonucleotides were purified for surface plasmon resonance measurements by reversed-phase HPLC (µBondapak C18, Waters). Those used for molecular biology techniques were purified by denaturing polyacrylamide gels. 3'-Biotinylated oligonucleotides containing a (64) photoproduct (T[64]T and CAAT[64]TAAG) were prepared as described previously (Kobayashi et al., 1998a).
Production and isolation of mutant 64M5 scFv's
Trp H33 was replaced by alanine, phenylalanine and tyrosine using PCR methods (Higuchi, 1989) and detailed procedures have been described previously (Kobayashi et al., 1999
). All mutant DNA sequences were confirmed by automated DNA sequencing (Applied Biosystems Model 373A). Once constructed, each mutant scFv gene was introduced into an expression plasmid in which the scFv was fused to a hexahistidine tag (Kobayashi et al., 1999
).
All of the scFv's accumulated in Escherichia coli cells in the form of inclusion bodies according to SDSPAGE and western blotting. Active proteins were obtained by solubilizing the inclusion bodies in 6 M guanidinium hydrochloride followed by an on-column refolding and purification procedure (Kobayashi et al., 1999). The scFv's were further purified by gel filtration using a Superose 12 HR 10/30 column equilibrated with HBS (10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% Tween-20) using a SMART-system (Pharmacia Biotech).
Surface plasmon resonance determination of (64) photoproduct binding by scFv's
Binding of scFv's to oligonucleotides containing a (64) photoproduct was measured by surface plasmon resonance (SPR) measurements using a BIAcore instrument as described previously (Kobayashi et al., 1999). The minimal amount of DNA was immobilized on the sensor chip in order to avoid mass transport limitations. Injections of biotinylated oligonucleotides (0.01 pmol/µl in HBS) were repeated until the SPR signal was increased by 1030 resonance units (RU) above the original baseline. Purified scFv's were diluted in HBS buffer, then they were injected over the immobilized oligonucleotides at a flow rate of 100 µl/min over a concentration range from 10 to 200 nM. Sensorgrams were recorded and normalized to a base line of 0 RU. Equivalent volumes of diluted antibodies were also injected over a non-oligonucleotide surface to serve as blank sensorgrams to allow subtraction of the bulk refractive index background. The association was monitored by measuring the rate of binding to antigen at different protein concentrations. The dissociation of these antibodies from the antigen surface was monitored after the end of the association phase. The remaining bound antibodies were removed completely by injecting 50100 µl 100 mM HCl. Kinetic rate constants were calculated using BIAevaluation 2.1 software (Biacore) using a single-site binding model (A + B = AB). The ratio of the rate constants allowed the apparent equilibrium constant to be calculated, KD,app = kdiss/kass.
Fluorescence measurements of (64) photoproduct with scFv's
Steady-state fluorescence excitation and emission spectra of (64) photoproduct with scFv's were measured on a Model FP-777 spectrofluorometer (Japan Spectroscopic Co, Ltd) using a 6 mm square cuvette and a sample volume of 200 µl. The emission spectra were recorded over the wavelength range from 250 to 500 nm with an excitation wavelength of 313 nm. The spectral bandpass was 5 nm for all emission spectra. After obtaining the emission scan for the photoproduct-containing oligonucleotides alone in a HBS buffer, scFv proteins were added at the indicated concentrations and allowed to equilibrate for 30 min at 25°C prior to spectral measurements.
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Results |
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We replaced Trp H33 by Ala, Phe and Tyr using standard site-directed mutagenesis techniques (Higuchi, 1989) and expressed the scFv proteins along with C-terminal hexahistidine tags in E.coli as inclusion bodies. The pure scFv proteins were prepared by solubilizing the inclusion bodies with denaturant, capturing the unfolded proteins on metal affinity columns, then re-folding the scFv proteins while still attached to the solid support (Kobayashi et al., 1999
). After elution from the metal affinity column, the scFv's were further purified by gel filtration chromatography. The final chromatography step also indicated that the wild-type and all three mutant scFv's were predominantly monomeric under the experimental conditions.
The rate constants for photoproduct binding by the scFv's were determined by surface plasmon resonance and the data are summarized in Table I. Two DNA's were used: the (64) photodimer and an octanucleotide that contained a central (64) photoproduct. The former was designed to probe interactions with the photoproduct itself while the latter would also allow contributions to binding energy by contacts with flanking nucleotides. The W-H33-A mutant was unable to bind the dimer at detectable levels under our experimental conditions, although binding to the octamer was measurable.
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Discussion |
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The alterations in (64) photoproduct fluorescence properties with changes in the H33 residue are also consistent with a -stacking interaction involving Trp H33. Each mutation was introduced into the X-ray crystal structure of the 64M5 scFv using computer modeling. Interestingly, the aromatic rings of both Tyr and Phe were predicted to overlap with the 3'-pyrimidone base more extensively than the indole side chain of Trp H33. While it is not possible to quantitatively interpret the fluorescence changes caused by the 64M5 scFv, we note that the decrease in (64) photoproduct fluorescence intensity caused by the wild type 64M5 scFv was identical to that observed for the proteolytically-prepared 64M5 Fab (data not shown), suggesting that this quenching is an intrinsic part of the binding interaction rather than an artifact of the scFv. However, we cannot determine whether these changes in fluorescence properties are due to changes in the relative angle between the pyrimidine and pyrimidone
-systems upon binding, resonance energy transfer to nearby
-systems or some other mechanism. It is clear that replacing Trp H33 with Phe or Ala alters the local environment of the (64) photodimer since binding is accompanied by large fluorescence increases that are not seen with the wild-type scFv.
In summary, the results of the present study argue that Trp H33 plays a key role in (64) photoproduct binding by the high-affinity 64M5 antibody. Whether enzymes that recognize and repair these photolesions will also utilize a similar interaction with the 3'-pyrimidone base remains to be determined.
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Acknowledgments |
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Notes |
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References |
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Received February 26, 1999; revised May 24, 1999; accepted June 21, 1999.