Crystal Structure of the Fab Fragment of an Anti-ofloxacin Antibody

Article pubs.acs.org/JAFC

Crystal Structure of the Fab Fragment of an Anti-ofloxacin Antibody and Exploration of Its Specific Binding Kuo He,†,‡ Xinjun Du,† Wei Sheng,† Xiaonan Zhou,† Junping Wang,† and Shuo Wang*,† †

Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Tianjin University of Science and Technology, Tianjin 300457, People’s Republic of China ‡ Hebei North University, Zhangjiakou, Hebei 075000, People’s Republic of China ABSTRACT: The limited knowledge on the mechanism of interactions between small contaminants and the corresponding antibodies greatly inhibits the development of enzyme-linked immunosorbent assay methods. In this study, the crystal structure of a Fab fragment specific for ofloxacin was obtained. On the basis of the crystal characteristics, the modeling of the interactions between ofloxacin and the Fab revealed that TYR31 and HIS99 of the heavy chain and MET20 and GLN79 of the light chain formed a hydrophobic region and that SER52 and ALA97 of the heavy chain and TYR35 of the light chain formed a salt bridge and two hydrogen bonds for specific binding. The key roles of SER52 and ALA97 were further confirmed by site-directed mutation. A specificity analysis using 14 ofloxacin analogues indicates that the length of the bond formed between the piperazine ring and the antibody plays key roles in specific recognition. This work helps to clarify the mechanisms through which antibodies recognize small molecules and improve immune detection methods. KEYWORDS: ofloxacin, Fab fragment, crystal structure, molecular recognition

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INTRODUCTION Ofloxacin (OFLX), an antibacterial drug that belongs to the fluoroquinolone class, has been widely used in the veterinary industry to treat and prevent various infectious diseases. Residues of drugs that enter the food chain and environment present various potential dangers to human health because they can lead to toxic effects, antibody-resistant strains of bacteria, and allergic reactions.1,2 For this reason, OFLX should be strictly monitored and controlled. In recent years, governmental agencies have set limitations on the acceptable levels of OFLX residues. Several traditional methods for the detection of OFLX residues, such as spectrophotometry,3−5 high-performance liquid chromatography (HPLC),6−8 capillary electrophoresis,9−11 and microbiological assay,12 are well-proven and widely accepted, but these methods are often viewed as laborious and time intensive for sample pretreatment. Moreover, implementation of these methods involves a significant investment in equipment. Immunoassays, which are based on antigen−antibody interactions, can avoid the drawbacks of chromatographic techniques and present high specificity, sensitivity, and simplicity. Moreover, immunoassays can be implemented at a low cost in a high-throughput configuration and are suitable for performing on-site analyses. This technique has been successfully developed to detect toxic compounds with low molecular weights, including pesticide residues, veterinary drug residues, environmental hormones, toxins, and prohibited food additives.13,14 The key step during the development of an immunoassay against small molecules is the production of an antibody with proper specificity and a high affinity. However, most immunoassays for the determination of small-molecule contaminants are still designed on the basis of “trial and error” as a result of the lack of understanding of the structural mechanisms of © XXXX American Chemical Society

hapten−antibody interactions. Haptens are small molecules that cannot elicit an immune response on their own; since the 1990s, some immunochemists have attempted to use molecular modeling to aid the rational design of haptens. These researchers designed several haptens as candidates and then used molecular modeling to optimize the energy and calculate the valences and charges. The hapten that was both structurally and electronically most similar to the target analyte was selected as the immunizing hapten.15−17 However, as a result of the limited knowledge of the structure of antibodies specific for small molecules, few studies have attempted to improve immunoassays by modifying the antibodies in a controllable manner, which greatly inhibits the development of immune detection methods. In the present study, the structure of an OFLX antibody was determined and the specific interactions between the antibody and its ligands were analyzed. The structural studies reported here provided insights into the hapten−antibody interaction and highlight the importance of understanding the atomic level mechanisms of the molecular recognition that occurs.

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MATERIALS AND METHODS

Materials and Instruments. The hybridoma cell line G10F5, which secretes a monoclonal antibody (mAb) against OFLX, was previously established in our laboratory. OFLX was provided by the China Institute of Veterinary Drug Control (Beijing, China). OFLX− ovalbumin (OVA) was conjugated in our laboratory. Goat anti-mouse (GaM) immunoglobulin G (IgG)−horseradish peroxidase (HRP) was obtained from Sigma-Aldrich Company (Shanghai, China). The Received: December 12, 2015 Revised: March 3, 2016 Accepted: March 10, 2016

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DOI: 10.1021/acs.jafc.5b05882 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

drawn by the PROCHECK program.27 All figures that presented structural representations were prepared with the PyMOL program.28,29 Atomic coordinates and experimental structure factors have been deposited in the PDB with the accession code 4Z0B. Modeling of the Interaction between the Fab Fragment and OFLX. The ligand was drawn with ChemOffice 2008.30 The geometry was optimized using the molecular dynamic method AMBER and the semi-empirical method PM3.31 The crystal structure of the Fab fragment was imported into an AutoDock workstation. All hydrogen atoms were added to the structure with their standard geometry; energy optimization was then performed using MOPAC7.0.32 The resulting model was subjected to a systematic conformational search at default parameters with a root-mean-square (RMS) gradient of 0.01 kcal/mol using Site Finder. A total of 80 cycles of calculation were used to obtain a final binding position that was as accurate as possible. The best conformation was selected on the basis of energetic grounds. The docking procedure was run, and the minimum final docking energy (ΔG) was calculated. Site-Directed Mutation and Validation. To evaluate the accuracy of the computational docking, two amino acids, namely, SER52 and ALA97, which provide the highest contributions to the binding, were mutated to alanine. Construction and expression of the Fab mutants were conducted as follows according to a previously described method using CHO cells.33 According to the sequence of the anti-OFLX Fab fragment, mutant cDNAs with enzyme digestion sites of heavy chain (HC) and light chain (LC) were synthesized (Genewiz, Beijing, China). Subsequently, the HC and pcDNA3 vector were digested with the EcoRI and Xba I restriction enzymes, and the LC and pcDNA3 vector were digested with the EcoRI and Xho I restriction enzymes. After the ligation reaction, a fragment containing a promoter, LC, and polyadenylation sequence was amplified from the pcDNA3−LC recombinant plasmid using primers with the BglII and Mun I enzyme digestion sites. The polymerase chain reaction (PCR)amplified products and the pcDNA3−HC plasmid were digested with the BglII and Mun I restriction enzymes, respectively. The digested products were purified with a PCR product purification kit and then ligated using the T4 DNA ligation enzyme. CHO cells were cultured to 85% confluence in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) at 37 °C under 5% CO2. The recombinant plasmid (pcDNA3−CH−CL) was transfected into CHO cells using the Lipofectamine 2000 transfection reagent (Thermo, Waltham, MA). All supernatants were collected, and the target Fabs were purified through protein L affinity chromatography (Thermo, Waltham, MA) according to the directions of the manufacturers. The binding activities of the mutant and wild-type Fab products were determined by ELISA as follows. Microplates were coated with 0.5 μg/well OFLX−OVA overnight at 37 °C and then washed twice with PBS containing 0.1% Tween-20 (PBST). The wells were then blocked with 0.5% skim milk. After the blocking buffer was discarded, the Fab was added to the wells and the plates were incubated at 37 °C for 1 h and then washed 5 times with PBST. A total of 50 μL of a solution of goat anti-mouse IgG-HRP (1:5000 in PBS) was then added to the wells. The enzyme reaction was subsequently performed with TMB as the substrate. After incubation for 20 min at room temperature, the reaction was terminated and the absorbance was measured at 450 nm. Basis for Specificity of Anti-OFLX mAb. A total of 14 OFLX analogues were selected to analyze the specificities of the mAb using the dc-ELISA method. The cross-reactivities of the OFLX mAb with other analogues were determined by comparing the average analogue concentrations required for 50% inhibition (IC50) to the value obtained for OFLX, which was run on the same plate. Simultaneously, the 14 analogues were docked with the Fab fragment crystal structure as described above, and the complex structures, key bonds, bond lengths, atoms, and residues involved in the interactions were obtained. The cross-reactivities, docking parameters, and analogue structures were combined to investigate the basis for the specificity of the anti-OFLX mAb.

absorbance values were read with a Multiskan Spectrum purchased from Thermo (Labsystems, Vantaa, Finland). Production and Characterization of OFLX mAb. Ascites fluid was produced in pristine-primed BALB/c mice by intraperitoneal injection of hybridoma cells. The fluid was collected 7−10 days after hybridoma injection, and the mAb was purified through protein A agarose affinity chromatography. A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was used to detect the activity of the mAb. The 96-well polystyrene ELISA plates were coated with 0.05 μg/well of OFLX mAb via incubation overnight at 4 °C. Coated plates were washed 3 times with PBST (0.01 mol/L phosphate-buffered saline containing 0.05% Tween-20 at pH 7.4), and the wells were blocked with 100 μL/well of 1% bovine serum albumin (BSA) for 1 h at 37 °C. Afterward, different concentrations of analyte standards (OFLX, in 50 μL volumes) in 10% (v/v) methanol in phosphatebuffered saline (PBS) and 50 μL of enzyme tracer OFLX−HRP solution (diluted 8000-fold in PBS) were added simultaneously to each well. After 1 h of incubation at 37 °C, the plates were washed and 100 μL/well of substrate solution [3′,5,5′-tetramethylbenzidine (TMB)] was added. After incubation for 20 min at room temperature, the reaction was terminated by 50 μL/well of 2 M H2SO4. Finally, the absorbance was measured at 450 nm. Fab Fragment Preparation and Purification. Fab fragments were prepared from whole anti-OFLX mAb by papain (Worthington, Lakewood, NJ) digestion. OFLX mAb (2−3 mg, concentration ranging from 1 to 2 mg/mL, in 20 mM phosphate buffer at pH 7.0) was digested with papain in the presence of L-cysteine and ethylenediaminetetraacetic acid (EDTA) for 4 h at 37 °C. The ratio of mAb/papain was 100:1 (w/w). The final concentrations of Lcysteine and EDTA were both 10 mM. The reaction was stopped by adding iodoacetamide to a final concentration of 20 mM. The Fab fragments were purified on a protein A column (Pierce, Waltham, MA), which binds to Fc fragments and undigested anti-OFLX mAb, whereas the Fab fragments were eluted into the flow through. The buffer of the purified Fab was thereafter changed to 25 mM Tris at pH 7.0 and concentrated to 10−12 mg/mL.18,19 Crystallization of the Fab Fragment. Fab fragment crystals were grown by the vapor diffusion method in hanging drops at 20 °C. Initial crystal screening was performed with nine kits: INDEX, CrystalScreen, SALT, SALTRX, PEGRX, and PEG/ION from Hampton Research (Aliso Viejo, CA) and PACT, JCSG+, and CUBICPHASE1 from QIAGEN (Dusseldorf, Germany). The protein drops contained 1 μL of Fab fragment (10 mg/mL) and 1 μL of reservoir solution. Finally, crystals of the Fab fragment were obtained from a reservoir solution comprising 0.2 M dibasic ammonium phosphate and 20% (w/v) polyethylene glycol (PEG) 3350 at pH 6.5. Crystals began to appear within 1 week and grew to a final size of 0.2 × 0.1 × 0.1 mm over a period of 7 months. X-ray Diffraction and Data Collection. Nylon loops were used to harvest the Fab fragment crystals, which were subsequently immersed in mother liquor supplemented with 15% glycerol for 1 min. Synchrotron data were collected on Area Detector Systems Corporation (ADSC) Q315 charge-coupled device (CCD) detectors (ADSC, Poway, CA) at the Shanghai Synchrotron Radiation Facility. Diffraction intensities were detected in half-degree images with an exposure time of 2 min/image. The X-ray data were processed using the HKL2000 program.20,21 Structure Solution and Refinement. The preliminary structure of the OFLX Fab fragment was solved by molecular replacement using the PHASER program.22,23 The Fab [Protein Data Bank (PDB) code 2QHR] that exhibited the highest sequence homology with OFLX Fab fragment was used as the search model. The PHENIX AutoBuild program was used for model building and obtaining improved maps.24,25 These maps were manually inspected, and the automatically built segments were rearranged to form a compact molecule, in which the missing segments were completed. PHENIX Refine was used to perform simulated annealing and to add solvent molecules to the model. Automated model building was subsequently performed with ARP/Warp. Finally, manual model building and adjustments were performed using the Coot program.26 The Ramachandran plots were B


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Figure 1. Sensitivity characterizations of anti-OFLX mAb and Fab fragment purification. (A) Normalized standard curve obtained by dc-ELISA of OFLX under optimized conditions. The error bars represent standard deviations from three repeated experiments, in which each inhibition with the same concentration of OFLX was performed in duplicate. (B) SDS−PAGE of the digested Fab fragment under reducing conditions after elution from a protein A−agarose affinity chromatography column. Lane M, protein marker; lane 1, Fab fragment.

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RESULTS Characterizations of Anti-OFLX mAb and Fab Fragment Preparation. To characterize the specificity and sensitivity of the purified anti-OFLX mAb, a dc-ELISA was performed. The sensitivity and detection limit (IC15) of the dcELISA (IC50) were 0.893 and 0.151 ng/mL, respectively (Figure 1A). The Fab of the anti-OFLX mAb was obtained after digestion with papain and purified using a protein A−agarose affinity chromatography column. Sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) was performed to detect the Fab segments (Figure 1B). The target Fab fragment has two obvious bands: the heavy chain at approximately 28 kDa and the light chain at approximately 26 kDa. The purity of the Fab fragment was sufficient for crystallization. Crystallization and Data Collection. After crystallization by vapor diffusion, the Fab fragments formed monoclinic crystals diffracting to 3.20 Å with one molecule in the asymmetric unit. The complete statistics of the synchrotron diffraction data are presented in Table 1. The crystal space group was assigned to C2. The unit cell had the following parameters: a = 62.842 Å, b = 53.204 Å, c = 129.157 Å, α = 90.000°, β = 98.819°, and γ = 90.000°. Matthew’s coefficient was 2.32 Å3/Da. Fab Fragment X-ray Crystal Structure. The structure was solved by molecular replacement and refined to 3.20 Å resolution. Final refinement provided crystallographic R and Rfree factors of 26.0 and 30.0%, respectively, with good stereochemistry (Table 1). The refined model includes 424 amino acid residues and 1 sulfate ion. The overall anti-OFLX Fab fragment crystal structure is shown in Figure 2A. Four typical domains [heavy-chain variable domain (VH), heavy-chain constant domain (CH), light-chain variable domain (VL), and light-chain constant domain (CL)] were found in the structure, and each of these contained two antiparallel β-sheets. The β-sheets of VH, VL, CH, and CL consist of 4, 5, 3, and 4 strands, respectively. Four disulfide bonds (between residues CYS27 and CYS87 of the light chain, between residues CYS133 and CYS193 of the light chain, between residues CYS22 and CYS96 of the heavy chain, and between residues CYS148 and CYS203 of the heavy chain) were observed in the Fab structure (Figure 2B). Residues 67, 69, 136, 139, and 141−142 of the heavy chain and residues 47,

Table 1. Data Collection and Refinement Statistics data collection wavelength (Å) space group cell dimensions (Å) angles (deg) resolution (Å) total number of reflections number of unique reflections completeness (%) average redundancy Rmergea I/σ(I) Wilson B factor (Å) solvent content (%) refinement resolution (Å) number of reflections rmsd bond lengths (Å) rmsd bond angles (deg) Rworkb (%) Rfreec (%) average B factors of protein Ramachandran plot (%) allowed generously allowed disallowed

1.5418 C2 a = 62.842, b = 53.204, and c = 129.157 α = 90.000, β = 98.819, and γ = 90.000 42.54−3.20 19826 6747 94.86 (96.68) 2.94 (2.85) 0.137 (0.41) 5.2201 (1.80) 27.6

50−3.20 (3.28−3.20) 6425 0.004 0.948 26.0 30.0 35

93.3 6.0 0.7

Rmerge = ∑∑i|I(h)i − ⟨I(h)⟩|/∑∑i|I(h)i|, where ⟨I(h)⟩ is the mean equivalent intensity. bRwork = ∑|Fo − Fc|/∑|Fo|, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively. c Rfree = ∑|Fo − Fc|/∑|Fo|. This value was calculated using a test data set comprising 5% of the total data that was randomly selected from the observed reflections. a

58, 88, and 96 of the light chain were not included in the model as a result of their weak and/or discontinuous electron densities. The distribution of complementarity determining regions (CDRs) comprising the combining site is shown in Figure 2C. H3 and L3 occupy the central part of the CDRs. H2 and L1 are located further out, and H1 and L2 are located on the periphery. The amino acid residues in the CDRs that have an C


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Figure 2. Crystal structure of the OFLX Fab fragment. (A) Ribbon representation of the overall OFLX Fab fragment structure. The four domains of the Fab and the variable (V) and constant (C) domains of the heavy (H) and light (L) chains are noted as VH (green), CH (pink), VL (sky blue), and CL (maroon), respectively. (B) Disulfide bonds on the OFLX Fab fragment. The four disulfide bonds are shown in yellow (1, CYS148− CYS203; 2, CYS27−CYS96; 3, CYS133−CYS193; and 4, CYS27−CYS87). (C) Variable regions of the anti-OFLX Fab fragment. The CDRs are shown inside the box (H1, H2, H3, L1, L2, and L3).

accessible surface area (ASA) of greater than 5 Å2 are listed in Table 2. The total ASA of each CDR was also calculated. H3

The CDR loops H2, H3, H1, and L3 bind to OFLX with good structural and chemical complementarity, as revealed by the shape complementarity value (Sc) of 0.58. A piperazine group containing a methyl group from OFLX stretches into the pocket on the Fab fragment, with a carboxyl terminal group pointing in the opposite direction toward the outside surface of the pocket. The protonated nitrogen atom (N3) on the piperazine group forms a salt bridge with the side chain of SER52 (a negatively charged carboxyl) from the heavy chain (Figure 3C). In addition to this strong polar bond, two hydrogen bonds are formed between OFLX and the anti-OFLX Fab fragment. One bond forms between O1 and ALA97 of the heavy chain, and the other bond forms between O2 and TYR35 of the light chain (Figure 3C). Furthermore, a total of 10 van der Waals contacts were observed between the anti-OFLX Fab fragment and OFLX (Figure 3D). CDR loops H1, H2, H3, and L3 form two (20%), one (30%), two (30%), and three (20%) van der Waals contacts with OFLX, respectively. Site-Directed Mutation and Validation. The expressed Fab products were purified by affinity chromatography and analyzed by SDS−PAGE. The results showed that the OFLX Fab and the mutated Fabs were obtained with high purity (Figure 4A). ELISA was performed to investigate the binding activity of the wild-type and mutated Fab antibodies to OFLX (Figure 4B). The results showed that the wild-type Fab exhibited high binding activity, whereas the two mutated Fab exhibited very weak binding activities to OFLX. This result demonstrates that the SER52 and ALA99 residues of Fab are critical for the specific binding of the antibody to OFLX. Basis for Specificity of the Anti-OFLX Antibody. To investigate the mechanism of specific interaction between OFLX and the antibody, 14 analogues were selected to perform dc-ELISA and docking analysis (Table 3). Among the 14 selected analogues, the cross-reactivity values of the mAb with 4

Table 2. ASA of the CDRs of the OFLX Fab Fragment CDR

sequence

total ASA (Å2)

H1 H2 H3 L1 L2 L3

SSYA YISTGGGST ARHNYYGGRIYSMDY ATM SGT QQGSENTET

130 236 504 42 64 436

contributes the largest ASA, at 504 Å2, followed by L3, which contributes an ASA of 436 Å2. The Kabat numbering scheme was used throughout. Interactions between the Fab Fragment and OFLX. Modeling of the binding complex revealed that the OFLX molecule binds the antigen recognition area of the anti-OFLX Fab fragment (Figure 3A). The hapten molecule generally binds at the interface of the heavy and light chains. The CDRL1 and CDR-L2 loops, which are located further from the interface, do not directly interact with OFLX. A small cavity in the antigen-binding site is formed by CDR-H1, CDR-H2, CDR-H3, and CDR-L3. Two key hydrogen bonds are formed among the CDRs (Figure 3B): CDR-H1 (TYR31) and CDRH3 (HIS99) participate in a hydrogen bond that helps form the active cavity, and CDR-L1 (MET20) and CDR-L3 (GLN79) form a hydrogen bond that maintains its stability. In contrast, the interactions among CDR-H1, CDR-H2, CDR-H3, and CDR-L3 occur via van der Waals contacts. Namely, the aromatic residues (TYR and TRP) of CDR-H2, CDR-L1, and CDR-L3 interact hydrophobically with each other, thus providing a hydrophobic region for the antigen-combining site. D


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Figure 3. Binding site of the anti-OFLX Fab fragment complexed with OFLX. (A) Space-filling representation of the complex between the ligand and the Fab fragment. CDR loops are shown in light pink (L1), sky blue (L2), light orange (L3), red (H1), green (H2), and yellow (H3). OFLX is presented in light brown. Frame regions for each chain are shown in blue. (B) Hydrogen bonds between CDR loops that form the OFLX-binding pocket (hydrogen bonds are shown as yellow dashed lines). (C) Three-dimensional representation showing selected amino acid residues. The salt bridge and hydrogen bonds are represented as yellow dashes. (D) Top view of the electrostatic potential surface of the Fab showing the large, deep pocket as the antigen-binding site with a hydrophobic periphery. The residues involved in the interaction are indicated.

Figure 4. Binding activity analysis of mutated Fab. (A) SDS−PAGE analysis of the expressed and purified Fab fragments. Lane 1, protein marker; lane 2, purified wild-type Fab; lane 3, protein marker; lane 4, purified SER52-mutated Fab; and lane 5, purified ALA97-mutated Fab. (B) Affinity assay. The affinities of three different Fabs to OFLX were determined by ELISA.

veterinary drugs were greater than 1%. Docking analysis indicated that the length of the salt bond formed between the antibody and the ligand was a key factor in determining the specific interaction. When the bond length was less than 2.5 Å,

the cross-reactivity value was at least 2.5%. For both cases in which the bond length was between 2.5 and 3.0 Å, the crossreactivity values were both 0.3%. However, if the length was greater than 3.0 Å, the cross-reactivity was less than 0.1% in all E


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Journal of Agricultural and Food Chemistry Table 3. Specificity Analysis of Analogues

anti-tetrodotoxin scFv antibodies using the same method. However, because this method is based on sequence homology, the information on antibody structures obtained by this method is limited and simplistic. Furthermore, distinct differences may exist among the results calculated by different software programs, which alludes to the substantial problem of the questionable accuracy of modeling methods exclusively based on sequence. In contrast, some researchers have attempted to investigate the binding mechanism of antibody−antigen pairs exclusively through analyses of the antigen structure without considering antibody structures. Mu et al.13 and Wang et al.37 explored the binding mechanisms of the anti-OFLX antibody

cases. In addition to the salt bond, hydrogen bonds formed between the antibody and the ligand appeared to play a role in influencing the specificity to a certain extent.

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DISCUSSION

Many recent studies have investigated the structures of antibodies to small-molecule contaminants and the mechanisms underlying the interaction between these antibodies and their ligands. Wen et al.34 obtained anti-OFLX scFv structures using a homology modeling method, and Wang et al.35 and Zheng and Lv36 simulated the structures of anti-deoxynivalenol and F


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instructive reference to design and synthesize efficient antibodies for other small molecules. In summary, the structure of the Fab antibody specific for OXFL was obtained in the present study. The key factors for determining the specificity of the antibody were also systematically analyzed. This work provides a foundation for understanding the mechanisms underlying the interactions between small molecules and their antibodies and for the preparation of antibodies with increased affinity and specificity.

from the antigen structure using a quantitative structure− activity relationship (QSAR). The results indicated that the piperazine ring signal affected the binding. Xu et al.15 and Yuan et al.38 investigated the binding mechanisms of anti-organophosphorus pesticide and anti-phenylurea herbicide antibodies using hologram quantitative structure−activity relationship (HQSAR) and QSAR models, respectively. The results showed that hydrophobicity notably affected the binding. On the basis of these results, it is clear that studies of the antigen structure alone cannot thoroughly elucidate the specific interactions between antibodies and their targets. Our work determined the three-dimensional structure of an anti-OFLX antibody through protein crystal and X-ray diffraction methods, providing details that greatly improve our understanding of the specific binding mechanisms. On the basis of the X-ray crystallographic analysis in combination with the results from our cross-reactivity and docking analyses, it is possible to speculate the basis for the specific interaction between OFLX and the antibody: (i) Similar to OFLX, R1 and R2 (substituent groups at sites 7 and 8 on the common quinolone structure) are key groups for specificity. Only atoms from these two groups can form polar contacts with the antibody. (ii) The presence of a piperazine ring at R1 is essential. If R1 is a piperazine ring, the N atom of the ring forms a polar contact with the antibody. If R1 is another group, this polar contact does not form. In addition, the methyl group of the piperazine ring is an important factor for specificity. The basis for this finding perhaps lies in the fact that a methyl group exhibits a hydrophobic effect on the N atom of the piperazine ring. (iii) The length of the salt bond formed between the antibody and the ligand is another key factor in determining the specificity of the interaction. As the length of the salt bond increases, the cross-reactivity value gradually decreases. Hydrogen bonds formed between the antibody and the ligand appear to influence the specificity to a lesser extent. (iv) Steric hindrance occasionally affects the interaction between the antibody and the ligand. This effect has been observed with marbofloxacin, enrofloxacin, and difloxacin, the R3 groups of that have been found to hinder ligand entrance into the cavity. To the best of our knowledge, this study provides the first report of the crystal structure of an antibody specific to a smallmolecule contaminant. Our work provides a reference for designing haptens and engineered antibodies in a controllable manner. In an efficient hapten design for quinolones, for example, the common structure of quinolones can be used as the parent molecule and different chemical groups, such as R1 and R2 from ZINC molecular databases, can be selected and installed on the parent. After numerous docking calculations using the crystal structure of the antibody, suitable new molecules will be sifted out, which will avoid the need for the random synthesis of different haptens and the subsequent evaluation of their immunogenicities. Furthermore, our work sets a useful reference for antibody design. Antibody modification is always untargeted, and numerous examinations must be performed to analyze the activities of the modified antibodies. Using the structure obtained in the current study as a template, numerous mutant antibodies can be designed. Subsequently, their activities can be evaluated using the key factors mentioned above for specific recognition, and their specificity can be selected simply via computer calculation and without real experimentation. It is reasonable to hypothesize that the key factors identified in this study can be used as an

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AUTHOR INFORMATION

Corresponding Author

*Telephone: 86-22-609112484. Fax: 86-22-60601332. E-mail: [email protected]. Funding

This work was supported by the National Science Foundation for Distinguished Young Scholars of China (Grant 31225021). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED OFLX, ofloxacin; BSA, bovine serum albumin; CDR, complementarity determining region; ELISA, enzyme-linked immunosorbent assay; mAb, monoclonal antibody; ASA, accessible surface area; TMB, 3′,5,5′-tetramethylbenzidine

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REFERENCES

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Crystal Structure of the Fab Fragment of an Anti-ofloxacin Antibody

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