A Human Antibody Fragment with High Affinity for Biodegradable

Mar 27, 2007 - Zinc Ion-binding Activity of an Anti-ZnO VHH Antibody, 4F2 ... Material Antibody Generation by Integrating Graft and Evolution Technolo...
0 downloads 0 Views 445KB Size
Download PDF
Bioconjugate Chem. 2007, 18, 645−651

645

A Human Antibody Fragment with High Affinity for Biodegradable Polymer Film Hideki Watanabe,† Kouhei Tsumoto,†,| Seiichi Taguchi,‡,⊥ Koichi Yamashita,‡ Yoshiharu Doi,‡ Yoshiyuki Nishimiya,§ Hidemasa Kondo,§ Mitsuo Umetsu,† and Izumi Kumagai*,† Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 6-6-11-606, Sendai 980-8579, Japan, Polymer Chemistry Laboratory, RIKEN Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan, and Functional Protein Research Group, Research Institute of Genome-based Biofactory (RIGB), National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira, Sapporo 062-8517, Japan. Received July 8, 2006; Revised Manuscript Received December 9, 2006

Antibodies with high affinity for the surface of a solid material would be advantageous in biomaterial science as a protein device. A human antibody fragment that binds to poly(hydroxybutyrate) (PHB), a biodegradable polymer matter, was generated by a phage display system. Clone PH7-3d3 was isolated after several rounds of selection and prepared as a fragment of immunoglobulin variable regions (Fv). The quartz crystal microbalance technique showed that PH7-3d3 Fv completely inhibited PHB enzymatic degradation by competing with PHB depolymerase. Kinetic analysis based on surface plasmon resonance demonstrated that PH7-3d3 Fv bound to the PHB film with an equilibrium dissociation constant of 14 nM. The three-dimensional structure of PH7-3d3 Fv was resolved to 1.7 Å, revealing that the complementarity determining regions (CDRs) in the Fv fragment form a relatively flat surface on which uncharged polar and aromatic amino acids are distributed in clusters. The structure of PH7-3d3 Fv was similar to that of PHB depolymerase in the orientation of aromatic residues in the binding sites. Alanine scanning mutagenesis demonstrated that these aromatic residues, especially tryptophan residues in CDRs, were critical in the interaction between PH7-3d3 Fv and PHB. Our results suggest the possible selection of an antibody fragment that binds a material surface in a manner similar to protein-ligand interaction.

INTRODUCTION Although most globular proteins target soluble molecules, some enzymes such as cellulose, chitinase, and PHB1 depolymerase recognize and bind to a solid-phase biopolymer. Many researchers in the interdisciplinary fields like nanobiotechnology have attempted to exploit these proteins as a specific molecule to solid phase, because they can specifically immobilize the molecules of interest on the support material (1, 2). For technological applications in which proteins serve as recognition devices, understanding the mechanism of recognition between solid surface and protein is necessary. To date, however, only limited information on the molecular recognition mechanism is available for three reasons: (i) few naturally occurring biomolecules that target solid surfaces are available, (ii) there are few efforts to generate such novel proteins de novo, and (iii) the methods for the analysis of the solid surface recognition mechanism are not well developed. The antibody, which plays a key role in the immune system, is a naturally occurring recognition device that shows excellent * Izumi Kumagai, tel: +81-22-795-7274; fax: +81-22-795-6164; [email protected]. † Tohoku University. ‡ RIKEN Institute. § AIST. | Present address: Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa 277-8562, Japan. ⊥ Present address: Division of Molecular Chemistry, Graduate School of Engineering, Hokkaido University, Sapporo 060-8628, Japan. 1 Abbreviations: Fv, fragment of immunoglobulin variable regions; VH, variable region of immunoglobulin heavy chain; VL, variable region of immunoglobulin light chain; CDR, complementarity determining region; QCM, quartz crystal microbalance; SPR, surface plasmon resonance; PHB, polyhydroxybutyrate; SBD, substrate binding domain.

binding ability and specificity. To our knowledge, although many antibodies against various antigens, including proteins, haptens, and carbohydrates, have been prepared by means of hybridoma technology, attempts to prepare the antibodies against solid surfaces have been unsuccessful, except the monoclonal antibody against amino acid crystals, because of the poor immunogenic potential of materials and the difficulty in sensitization of the vertebrate immune system by materials. In contrast, immunization is not required in antibody production based on the in vitro selection method such as the phage display system, thus allowing the preparation of antibodies that recognizes various material surfaces. Although some peptides bound to solid surfaces such as gold (4), GaAs (5), and ZnS (6) were selected using phage display (1, 7, 8), few studies involve the binding of an antibody to a solid surface, which is capable of showing higher affinity compared with a low molecular weight peptide and being a model for studying the mechanism of molecular recognition between a material surface and a biomacromolecule. The aim of the present study is to select a human antibody fragment that binds to a solid surface by phage display and to analyze the mechanism of the interaction between the antibody and the solid surface in detail. We use poly(hydroxybutyrate) (PHB), a biodegradable polymer, as a model material surface. PHB, which was found as an insoluble granule in bacteria (9), is a polyester of hydroxybutyric acid. The PHB granule serves as a reservoir of non-nitrogenous material under the conditions of insufficient nourishment; therefore, the host bacteria also carry the catabolic enzyme PHB depolymerase. PHB granules are produced in many bacteria, including Bacillus megaterium and Alcaligenes eutrophus, but also on the plasma membranes of Escherichia coli (10) and human erythrocytes (11). From a practical point of view, the effective combination of a human antibody fragment and a naturally occurring biomaterial would lead to material and medical engineering applications as well

10.1021/bc060203y CCC: $37.00 © 2007 American Chemical Society Published on Web 03/27/2007

646 Bioconjugate Chem., Vol. 18, No. 3, 2007

as providing a model for studying specific interactions between proteins and solid-phase surfaces.

EXPERIMENTAL PROCEDURES Selection of Antibody Fragments. Human antibody fragment libraries were subjected to selection on the basis of Fv fragment stabilization under coexistent antigens as described previously (12). Variable regions of light and heavy chains (VL and VH, respectively) were displayed on the filamentous bacteriophage M13. The phage viruses displaying VL chain with affinity for PHB, were first selected from the VL chain library, and then the selection and assay of the phage virus with the VH chain was carried out in the presence of the selected VL chain for stabilizing the VH chain. PHB purchased from ICI, Inc., was dissolved in chloroform and then dispersed in an amount of hexane to form refined PHB precipitates. Approximately 109 phages were mixed with 3 mg of the prepared PHB matter in 1 mL of PBS and 0.1% Tween 20 detergent. The mixture was incubated at room temperature with gentle agitation for 1 h. The residual phage viruses on PHB matter were amplified by directly adding the PHB matter into E. coli media after the exclusion of the unbound phage from the phage-PHB solution. After three rounds of selection, random clones were isolated, expressed as soluble fragments, and tested in enzyme-linked immunosorbent assay (ELISA). ELISA Analysis. PHB was adsorbed on 96-well microtiter plates. Antibody fragments were applied to the plates after nonspecific binding to the plates was blocked by using Super Blocking Buffer (Pierce), and the plates were incubated for 1 h at room temperature. After five washes with PBS, the binding of antibody fragments was detected with Fluoroskan Ascent FL (Labsystems), using ECL Western Blotting Detection Reagent (Amersham Biosciences). Preparation of Antibody Fragments. The VH and VL genes were inserted into the secretory coexpression vector pRA2; the resulting plasmid was named pRA2FHL. E. coli strain BL21 (DE3) was transformed with pRA2FHL and cultured at 28 °C for 18 h in 2 × YT medium containing 100 µg/mL ampicillin. Expression of the Fv fragment under the control of the T7 promoter was induced by adding 1 mM isopropyl β-Dthiogalactopyranoside (IPTG). The harvested cells were centrifuged and suspended in 50 mM Tris-HCl (pH 8.0) buffer with 200 mM NaCl. After sonication, the suspension was centrifuged at 5800 g for 30 min at 4 °C. The gene products in the supernatant were refined by means of a metal-chelate chromatography column that interacted with the histidine tag in the expressed VH and VL, after salting out with ammonium sulfate. Quartz Crystal Microbalance (QCM) Analysis. The apparatus and experimental procedure for QCM analysis were essentially the same as those described previously (13). The QCM oscillator in a QCA-917 (SEIKO EG&G) was washed with a freshly prepared piranha solution (H2SO4/H2O2 ) 3/1, v/v), and it was rinsed with distilled water several times. Dissolved PHB in chloroform (1.5 wt %) was cast on one side of the oscillator placed on a spin-coater (MOC Co., Ltd., ME300) at 4000 rpm under dry air, and then the film was kept at 110 °C for 1 day after the melting treatment at 210 °C for 30 s on a hot stage (Linkam LK-600PM). After stabilization of QCM in a 0.01 M phosphate buffer solution (pH 7.4) for 1 day, the selected Fv fragment was injected into the QCM cell. For the inhibition assay for PHB enzymatic degradation, the selected Fv fragment was injected 4.5 h after the replacement from an enzyme-free solution to a 0.02 µM enzyme solution. Surface Plasmon Resonance (SPR) Analysis. For the SPR analysis, a sensor chip of Au (Biacore) was ultrasonically cleaned in acetone, soaked in a freshly prepared piranha solution,

Watanabe et al. Table 1. Adsorption Equilibrium Constants for PH7-3d3 and Naturally Occurring Depolymerasesa protein

substrate

Ke [mL/µg]

organism

PH7-3d3 SBDCac SBDCte SBDAfa SBDI SBDII SBDII-I CenA Cex EndoIII E3 CBHI

PHB PHB PHB PHB PHB PHB PHB cellulose (BMCC) cellulose (BMCC) cellulose (Avicel) cellulose (Avicel) cellulose (Avicel)

2.9 1.0 1.1 0.8 0.26 0.07 0.4 0.041 0.033 0.012 0.0003 0.00054

human Comamonas acidoVorans (18) Comamonas testosterone (18) Alcaligenes faecalis (18) Pseudomonas stutzeri (19) Pseudomonas stutzeri (19) Pseudomonas stutzeri (19) Cellulomonas fimi (35, 39) Cellulomonas fimi (35, 39) Trichoderma Viride (36, 39) Thermobifida fusca (37, 39) Trichoderma reesei (38, 39)

a The K value of PH7-3d3 was calculated from the K determined by e D SPR analysis. Reported values for substrate binding domain (SBD) of PHB depolymerases and for cellulases were listed as references.

Table 2. Crystallographic Data for PH7-3d3 Fv space group unit cell dimensions VM (Å3) wavelength (Å) resolution (Å) completeness (%) resolution range R factor free R factor

I222 a ) 63.4, b ) 73.6, c ) 103.3 2.4 (Z ) 8) 0.979 1.7 99.3 20-1.7 19.0 21.4

and rinsed with double-distilled water in advance. PHB dissolved in chloroform (0.25% w/v) was spin-cast onto the chip at 6000 rpm. The chip was melted at 210 °C for 30 s and then kept at 110 °C for 24 h. The SPR resonance angle was confirmed to be within the range needed to detect binding. SPR measurements were performed using Biacore 2000 (Biacore). The running buffer for the experiments was PBS. Kinetic parameters were determined on the basis of a 1:1 Langmuir model by using the data from association phases. X-ray Crystallography. The soluble Fv was concentrated to 10 mg/mL by using Centricon YM-10 (Millipore). Fv was crystallized in 20% PEG 8000, 100 mM CHES (pH ) 9.3). Diffraction data to 1.7 Å were collected at 100 K by using synchrotron radiation on beamline 6A of the Photon Factory (Tsukuba, Japan). The diffraction images were integrated with the hkl program DENZO, and the intensity data were processed with SCALA in the CCP4 suite (14). The crystallographic data and statistics are summarized in Table 2. The structure was determined by a molecular replacement method with the program Molrep (15) in the CCP4 suite. The coordinates were refined using the programs REFMAC (16) and CNS (17). The atomic coordinates and structure factors were deposited in the Protein Data Bank (entry code 2D7T). Alanine Scanning Mutagenesis. The solvent-exposed amino acid residues in the complementarity determining regions (CDRs) were chosen for mutagenesis analysis. Each site-directed Fv mutant in which an amino acid in CDRs was mutated to alanine residue was performed by overlap extension PCR (Supporting Information). All the Fv mutants were prepared as described in the section of preparation of antibody fragments, and their binding to PHB surfaces were analyzed using SPR.

RESULTS Selection of a Human Antibody Fragment on PHB Surface. Using a phage-displayed antibody library, the antibody fragments with affinity for PHB were selected. After three rounds of selection, ELISA was used to test the randomly isolated clones for their binding to PHB (Figure 1A). Clone PH7-3d3 showed tight binding to PHB as indicated by its high

Human Anti-Plastic Antibody

Bioconjugate Chem., Vol. 18, No. 3, 2007 647

Figure 2. QCM analysis for the binding of Fv to PHB. A. Dependency of dose on the concentration of the injected Fv. B. PHB enzymatic degradation in the presence (red) and absence (blue) of PH7-3d3 Fv. Arrows indicate the injection of PHB depolymerase and Fv.

Figure 1. Screening of antibody fragments bound to PHB. A. ELISA analysis of VH clones selected after the three rounds of selection. The binding of clones was detected as chemiluminescence intensity. The inset shows structural formula of poly(hydroxybutyrate) (PHB). B. The CDR amino acid sequences of the clone PH7-3d3.

chemiluminescence value. The amino acid sequences of the complementary determining regions (CDRs) of the selected clone PH7-3d3 are shown in Figure 1B. The tip of the CDRH3 loop is composed of four amino acid residuessGly, Trp, Trp, and Glysthat lie in series, which seems to be the distinctive feature of CDR-H3. The roles of these residues are discussed later. For further analysis, the clone PH7-3d3 was prepared as soluble Fv. SDS polyacrylamide gel electrophoresis verified the purity of samples and confirmed that both chains of the Fv existed in a 1:1 molar ratio. The final yield of Fv was 40 mg per 1 L culture of E. coli (data not shown). QCM Analysis. We assessed the ability of PH7-3d3 Fv to bind to a PHB film by using the QCM technique (13). QCM analysis showed that the PH7-3d3 Fv bound tightly to the PHB film, and its binding was detected at the concentration of approximately 40 nM (Figure 2A). We also measured the QCM sensorgram of other Fv fragment with higher chemiluminescence

in ELISA assay than that of the background; however, no frequency changes were observed (data not shown), indicating that only the clone PH7-3d3 has the binding function to the PHB film. PHB, which is derived from storage materials of bacteria, can be hydrolyzed by PHB depolymerase. Using QCM, we then analyzed how PH7-3d3 Fv altered the biodegradability catalyzed by PHB depolymerase derived from Alcaligenes faecalis T1 (Figure 2B). A frequency shift corresponds to a mass change caused by PHB hydrolysis. Enzymatic hydrolysis of PHB film by the depolymerase was completely inhibited by the addition of the Fv (Figure 2B, arrow). A rapid frequency decrease of 80 Hz occurred on the injection of PH7-3d3 Fv, and then the value of this frequency change is nearly equivalent to the shift caused by the Fv binding. The inhibition of hydrolysis is, therefore, attributed to the specific coating of the Fv onto the PHB surface. Kinetic Study Based on Surface Plasmon Resonance (SPR) Analysis. We then analyzed the binding of PH7-3d3 Fv to the spin-coated PHB film by SPR (Figure 3A). The binding of PH73d3 Fv was confirmed, whereas there were no noteworthy responses upon the addition of hen egg-white lysozyme (HEL) and anti-HEL antibody HyHEL10 Fv to the PHB film. PHB, as seen from the structural formula, contains more oxygen atoms than other hydrophobic polymers such as polystyrene, and it is assumed to hydrophobically adsorb less globular protein. The binding of PH7-3d3 Fv to the dextran layer was not detected (Figure 3A). To examine the recognition mechanism in detail, quantitative analysis from the aspect of kinetics is necessary. Although the kinetic studies based on SPR have been performed for proteinprotein interaction to gain insight at the molecular level, we have obtained limited information regarding protein-material surface interaction to date. This difference may be due to protein denaturation on the surface, which influences the dissociation of adsorbed proteins and complicates the fitting analysis using a particular model. Here, we measured the binding affinity of

648 Bioconjugate Chem., Vol. 18, No. 3, 2007

Figure 3. SPR analysis of the binding of PH7-3d3 to the PHB surface. A. Binding behavior of several kinds of protein on the PHB surface. Hen egg-white lysozyme (HEL) and the anti-HEL antibody fragment HyHEL10 Fv were used as control proteins. The binding of PH7-3d3 Fv to a layer of dextran also is shown. The concentration of all samples used in the observation was 200 nM. B. dR/dt vs R plots of the association and dissociation (inset) phases. C. Dose dependency of dR/dt vs R plots. D. The apparent association rate constant (kobs) vs concentration plots. The association rate constant (kon) and dissociation rate constant (koff) were determined from the slope and intercept of the line. The value of the dissociation constant (KD) was calculated as koff/kon.

PH7-3d3 Fv to the PHB surface according to a 1:1 Langmuir model using only the association phase. As shown in Figure

Watanabe et al.

3B, dR/dt versus R plots for the dissociation phase did not yield a straight line, whereas the association phase showed good agreement with the model. Figure 3D shows a plot of the apparent association rate constant, kobs, versus concentration and the line’s slope and intercept, which are equal to kon and koff, respectively. From these results, the kinetic parameters kon, koff, and KD were determined to be 1.2 × 105 s-1 M-1, 1.7 × 10-3 s-1, and 1.4 × 10-8 M, respectively. The adsorption equilibrium constant of PH7-3d3 was calculated to be Ke ) 2.9 mL/µg, which is comparable to or larger than those of the substrate binding domains (SBD) of PHB depolymerases as reported by Kasuya et al. (18) and Ohura et al. (19). In comparison to other depolymerases including cellulases, PH7-3d3 Fv showed a substantially higher affinity for the substrate (Table 1). These results indicate the possibility that in vitro selection enables us to generate human antibody fragments with higher affinities than those of the naturally occurring substrate binding domains. X-ray Crystallography. Little information on the mechanism of molecular recognition between protein and the material surface is available at the amino acid level. To date, molecular dynamics (MD) simulation or docking studies have been performed for the interactions between solid surfaces and gold binding protein (20), carbon nanotube binding peptide (21), and amino acid crystal binding antibody (22). To gain insight into the interaction between PH7-3d3 and the PHB film surface, we performed X-ray structural analysis of PH7-3d3 Fv. Structural determination of PH7-3d3 Fv was based on the molecular replacement method using various search models (pdb codes 1AD9 for VH, 1DEE for VL) and was refined to an R value of 19.0% (Rfree ) 21.4%). Figure 4A shows the overall structure of the Fv. The CDR loops, which in general build an antigen binding site, formed a relatively flat surface that resembled those of antibodies against macromolecular antigens like proteins (23). Note that the putative antigen binding site lacks a cavity or groove, which typically is seen in those of antibodies for low molecular weight haptens and peptide or carbohydrate antigens (23). Although we have not obtained the structural data for any anti-plastic antibodies other than PH7-3d3, a flat binding site is likely to generate a highly complementary binding interface with the planar polymer surface. In addition, we examined the amino acid residues in the binding site for PHB. Polar amino acid residues such as Asn are juxtaposed around a cluster of aromatic amino acid residues (Trp and Tyr). Using ICM-REBEL (Molsoft), we calculated the electrostatic potential of the molecular surface around the CDRs (Figure 4B). The molecular surface formed from the CDRs is mainly a noncharged polar surface, which we anticipate is favored in the binding to uncharged plastic. Alanine Scanning Mutagenesis. On the basis of the threedimensional structure of PH7-3d3 Fv, we used alanine scanning mutagenesis to identify the amino acid residues critical for interaction. All the solvent-exposed amino acid residues in the CDRs were chosen as target residues for mutation (Figure 4B). We determined the equilibrium dissociation constant (KD) of the mutants by SPR, and then their Gibbs free energy changes were calculated. The involvement of solvent-exposed residues in antigen binding was represented as relative Gibbs energy changes (Figure 5). Each loss of the binding free energy (∆∆G) due to the point mutation was illustrated in color on a spacefilling model of Fv (Figure 4C). Figure 5 shows that the mutation of aromatic residues to alanine residue tended to affect the interaction more than did the mutation of polar residues. Of all the solvent-exposed residues in the CDRs, the mutation of HN32, HW50, HW100, HW101, and LH92, which are localized in the center of the

Human Anti-Plastic Antibody

Bioconjugate Chem., Vol. 18, No. 3, 2007 649

Figure 4. Structural analysis of PH7-3d3 Fv. A. Molecular surface as viewed from the side. The VH domain is shown in yellow; VL is in green. The CDR loops are colored in red in both chains. B. The molecular surface around the CDRs, as viewed from the top. Solvent-exposed amino acid residues in CDRs are annotated. C. Mapping of the amino acid residues critical for the binding of PH7-3d3 to the PHB surface. The resulting decrease in the binding due to alanine substitution was converted to ∆∆G (see Figure 5). The value of ∆∆G in each solvent-exposed residue is color-coded on the model.

Figure 5. The relative Gibbs free energy changes of the alaninesubstituted mutants. The equilibrium dissociation constants (KD) of each mutant were determined by the same method described in the case of wild type (WT). The Gibbs free energy ∆G was calculated by the equation ∆G ) RT ln KD. The change in the Gibbs free energy ∆∆G of each mutant was obtained by the subtraction of ∆G of wild type from those of mutants.

CDRs (Figure 4B), resulted in the marked reduction of affinity. Note that the two tryptophan residues HW100 and HW101, which are adjacent to each other in the CDR-H3 loop, have pronounced effects on binding. Tryptophan residues are known to frequently occur in the binding site critical for protein-protein interactions (24). To assess the roles of the tryptophan residues, we made three mutantssHW100F, HW101F, and HW100FW101Fswhere the tryptophan residues were mutated to phenylalanine. Interestingly, all three mutants bound to the PHB surface with nearly equal affinity (Figure 5), indicating that the binding tolerates the mutation of tryptophan to another aromatic residue. Three amino acid residuessHN32, HW50, and LH92s were also shown to affect the binding to PHB film. In comparison with HW50 and LH92, the side chain of HN32 is buried and seems to be unable to contact the antigen. The location of HN32 beneath HW100 suggests that HN32 might sustain the orientation of HW100 and stabilize the conformation of the CDR H3 loop to optimally allow it to contact the antigen.

DISCUSSION PHB, which has been developed as a biodegradable material, is a naturally occurring plastic, and its depolymerases have been isolated from several kinds of bacteria (18, 19). We considered that the comparison of the naturally occurring depolymerases with the in vitro selected antibody and the analogical approach

to the antibody-antigen interaction mechanism would contribute to the analysis of the protein-material surface interaction. Yamashita et al. analyzed protein adsorption to PHB film and used QCM to analyze the hydrolysis mechanism (25). They reported that PHB hydrolysis was not inhibited by the addition of bovine serum albumin or cellulase, which showed minimal binding of the proteins to PHB. In contrast, a PHB depolymerase mutant entirely lacking the catalytic site completely blocked PHB biodegradation. Therefore, the authors suggested that specific interactions were needed for biodegradation inhibition caused by the exchange of depolymerase for other proteins. Exchange of proteins on the surface depends not only on the binding site, but also on the binding affinity. Our SPR analysis revealed that PH7-3d3 Fv bound to the PHB surface with higher affinity than PHB depolymerase (Table 1). Therefore, PH7-3d3 Fv is capable of competing with PHB depolymerase, which was also supported by QCM assay (Figure 2B). Kasuya et al. aligned amino acid sequences of substrate binding domains in PHB depolymerases derived from Alcaligenes faecalis T1, Comamonas testosteroni, Comamonas sp., Streptomyces exfoliates, Psuedomonas pickettii, and Pseudomonas lemoignei (18). They identified tyrosine and phenylalanine (aromatic residues) and serine, threonine, glutamine, and asparagine (polar residues) as conserved residues in the depolymerases. The driving force for the binding to the PHB surface is not electrically charged, but may be hydrogen bonding, van der Waals contact, or hydrophobic effect; therefore, it is reasonable to consider that all of these residues may be involved in binding. Except for phenylalanine, which does not occur in the tip of CDR loops frequently according to germline gene sequence (26), all of the listed residues appear in the CDRs. The crystal structure of PHB depolymerase from Penicillium funiculosum was recently determined by Hisano et al. (27). The comparison of the structure of PH7-3d3 Fv with that of the depolymerase revealed that they had some similarities, especially in the distribution of solvent-exposed amino acid residues on the putative binding sites. In the two models, a number of hydrophobic residues on the molecular surface, which contribute to the adsorption to the PHB surface via hydrophobic interaction, and some polar residues around them were aligned in a plane. Some tyrosine and tryptophan residues located in the antigen binding site of PH7-3d3 and the tyrosine residues in the PHB adsorption site of the depolymerase had the same orientation in a way that allowed the aromatic rings to be in face-to-face contact with the PHB surface. The orientation could be effective in adsorption to the surface. The sequential and structural similarities suggest that PH7-3d3 Fv and PHB depolymerases might bind to PHB surface in a similar manner, which is consistent with the results of kinetic analysis and inhibition assay using SPR and QCM.

650 Bioconjugate Chem., Vol. 18, No. 3, 2007

The alanine scanning mutagenesis studies showed that HN32, HW50, LH92, and especially HW100 and HW101, were critical for the interaction between PH7-3d3 Fv and the PHB film (Figure 5). On the other hand, mutants HW100F, HW101F, and HW100FHW101F, in which tryptophan residues in CDR-H3 were mutated to phenylalanine, have no decrease of binding affinity (Figure 5). This finding indicates that the aromatic ring can compensate for the key interaction between PH7-3d3 and PHB film via the indole ring of Trp, and it strongly suggests that the driving force of the binding is not due to hydrogen bonding but results from other factors such as van der Waals contacts, π electron donors, or hydrophobic effect. Complementarity at the molecular interface becomes particularly important when binding depends on these factors. The planar molecular surface formed by the CDRs of PH7-3d3 Fv suggests that the deletion of the bulky aromatic ring leads to decreased complementarity between binding interfaces. Bogan et al. statistically identified tryptophan, tyrosine, and arginine as the residues that are apt to be “hot spots” (i.e., that various specific residues play a key role in protein-protein interaction) (24). In contrast, the mutation of polar residues surrounding these aromatic residues had less influence on binding. They subsequently proposed a mechanism in which the residues in the center of the binding surface are important as hot spots, and this role originates from being shielded from solvent by the surrounding polar residues. In the case in which those polar residues work as a solvent-shielding ring (referred to as the “O-ring”), single mutation is incapable of decreasing the binding ability. Then, our results (Figure 4) can be discussed in analogy with the concept of hot spots that has been applied to a number of protein-protein interactions, suggesting the possibility of in vitro selection of an antibody that recognizes a material surface in a similar manner to protein-ligand interaction.

CONCLUSION Few works regarding the proteins that function at the solidliquid interface have been reported, because only a few natural examples of these kinds of proteins have ever been identified and characterized. The specific mechanism underlying the interaction between a protein and a material surface remains to be revealed. In this study, we focused on a natural antibody repertoire and selected an antibody fragment that binds to PHB from a human antibody library. This is the first report of generating a human antibody fragment with high affinity for a given material surface based on phage display system. Protein engineering of antibodies (i.e., antibody engineering) has generated vast amounts of information and knowledge, including that of macro- and microscopic interaction analysis (28-31), improvement of functional properties based on site-directed mutagenesis analysis (32) or evolutionary engineering approaches (33), and construction of multivalent antibody fragments (34). The methodology we show here links the knowledge of material engineering with that of antibody engineering. From this practical viewpoint, our current work can be viewed as the generation of a highly regulated protein device that functions on the surface of a material and leads to self-assembly of materials via antibodies targeting solid surfaces.

ACKNOWLEDGMENT We thank Dr. K. Makabe for helpful discussions. This work was supported in part by the Grant-in-aid for Scientific Research (S) from JSPS, Japan. Supporting Information Available: The list of the oligonucleotide primer pairs for the alanine scanning mutagenesis of Fv

Watanabe et al.

fragment. This material is available free of charge via the Internet at http://pubs.acs.org.

LITERATURE CITED (1) Sarikaya, M., Tamerler, C., Jen, A. K., Schulten, K., and Baneyx, F. (2003) Molecular biomimetics: nanotechnology through biology. Nat. Mater. 2, 577-585. (2) Ji, Q., Vincken, J. P., Suurs, L. C., and Visser, R. G. (2003) Microbial starch-binding domains as a tool for targeting proteins to granules during starch biosynthesis. Plant Mol. Biol. 51, 789-801. (3) Geva, M., Frolow, F., Eisenstein, M., and Addadi, L. (2003) Antibody recognition of chiral surfaces. Enantiomorphous crystals of leucine-leucine-tyrosine. J. Am. Chem. Soc. 125, 696-704. (4) Brown, S. (1997) Metal-recognition by repeating polypeptides. Nat. Biotechnol. 15, 269-272. (5) Whaley, S. R., English, D. S., Hu, E. L., Barbara, P. F., and Belcher, A. M. (2000) Selection of peptides with semiconductor binding specificity for directed nanocrystal assembly. Nature (London) 405, 665-668. (6) Lee, S. W., Mao, C., Flynn, C. E., and Belcher, A. M. (2002) Ordering of quantum dots using genetically engineered viruses. Science 296, 892-895. (7) Naik, R. R., Stringer, S. J., Agarwal, G., Jones, S. E., and Stone, M. O. (2002) Biomimetic synthesis and patterning of silver nanoparticles. Nat. Mater. 1, 169-172. (8) Wang, S., Humphreys, E. S., Chung, S. Y., Delduco, D. F., Lustig, S. R., Wang, H., Parker, K. N., Rizzo, N. W., Subramoney, S., Chiang, Y. M., and Jagota, A. (2003) Peptides with selective affinity for carbon nanotubes. Nat. Mater. 2, 196-200. (9) Lemoigne, M. (1927) Microbial autolysis and the autolytic origin of β-hydroxybutyric acid. Ann. Inst. Pasteur 41, 148. (10) Huang, R., and Reusch, R. N. (1996) Poly(3-hydroxybutyrate) is associated with specific proteins in the cytoplasm and membranes of Escherichia coli. J. Biol. Chem. 271, 22196-22202. (11) Reusch, R. N., Huang, R., and Kosk-Kosicka, D. (1997) Novel components and enzymatic activities of the human erythrocyte plasma membrane calcium pump. FEBS Lett. 412, 592-596. (12) Watanabe, H., Tsumoto, K., Asano, R., Nishimiya, Y., and Kumagai, I. (2002) Selection of human antibody fragments on the basis of stabilization of the variable domain in the presence of target antigens. Biochem. Biophys. Res. Commun. 295, 31-36. (13) Yamashita, K., Aoyagi, Y., Abe, H., and Doi, Y. (2001) Analysis of adsorption function of polyhydroxybutyrate depolymerase from Alcaligenes faecalis T1 by using a quartz crystal microbalance. Biomacromolecules 2, 25-28. (14) Collaborative Computational Project, No. 4. (1994) The CCP4 suite: programs for protein crystallography. Acta Crystallogr., Sect. D: Biol. Crystallogr. 50, 760-763. (15) Vagin, A., and Teplyakov, A. (1997) MOLREP: an automated program for molecular replacement. J. Appl. Cryst. 30, 1022-1025. (16) Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr., Sect. D: Biol. Crystallogr. 53, 240255. (17) Bru¨nger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr., Sect. D: Biol. Crystallogr. 54, 905-921. (18) Kasuya, K., Ohura, T., Masuda, K., and Doi, Y. (1999) Substrate and binding specificities of bacterial polyhydroxybutyrate depolymerases. Int. J. Biol. Macromol. 24, 329-336. (19) Ohura, T., Kasuya, K. I., and Doi, Y. (1999) Cloning and characterization of the polyhydroxybutyrate depolymerase gene of Pseudomonas stutzeri and analysis of the function of substratebinding domains. Appl. EnViron. Microbiol. 65, 189-197. (20) Braun, R., Sarikaya, M., and Schulten, K. (2002) Genetically engineered gold-binding polypeptides: structure prediction and molecular dynamics. J. Biomater. Sci., Polym. Ed. 13, 747-757. (21) Wang, S., Humphreys, E. S., Chung, S. Y., Delduco, D. F., Lustig, S. R., Wang, H., Parker, K. N., Rizzo, N. W., Subramoney, S., Chiang, Y. M., and Jagota, A. (2003) Peptides with selective affinity for carbon nanotubes. Nat. Mater. 2, 196-200.

Bioconjugate Chem., Vol. 18, No. 3, 2007 651

Human Anti-Plastic Antibody (22) Geva, M., Eisenstein, M., and Addadi, L. (2004) Antibody recognition of chiral surfaces. Structural models of antibody complexes with leucine-leucine-tyrosine crystal surfaces. Proteins 55, 862-873. (23) MacCallum, R. M., Martin, A. C., and Thornton, J. M. (1996) Antibody-antigen interactions: contact analysis and binding site topography. J. Mol. Biol. 262, 732-745. (24) Bogan, A. A., and Thorn, K. S. (1998) Anatomy of hot spots in protein interfaces. J. Mol. Biol. 280, 1-9. (25) Yamashita, K., Funato, T., Suzuki, Y., Teramachi, S., and Doi, Y. (2003) Characteristic interactions between poly(hydroxybutyrate) depolymerase and poly[(R)-3-hydroxybutyrate] film studied by a quartz crystal microbalance. Macromol. Biosci. 3, 694-702. (26) Zemlin, M., Klinger, M., Link, J., Zemlin, C., Bauer, K., Engler, J. A., Schroeder, H. W., Jr., and Kirkham, P. M. (2003) Expressed murine and human CDR-H3 intervals of equal length exhibit distinct repertoires that differ in their amino acid composition and predicted range of structures. J. Mol. Biol. 334, 733-749. (27) Hisano, T., Kasuya, K., Tezuka, Y., Ishii, N., Kobayashi, T., Shiraki, M., Oroudjev, E., Hansma, H., Iwata, T., Doi, Y., Saito, T., and Miki, K. (2006) The crystal structure of polyhydroxybutyrate depolymerase from Penicillium funiculosum provides insights into the recognition and degradation of biopolyesters. J. Mol. Biol. 356, 993-1004. (28) Braden, B. C., Goldman, E. R., Mariuzza, R. A., and Poljak, R. J. (1998) Anatomy of an antibody molecule: structure, kinetics, thermodynamics and mutational studies of the antilysozyme antibody D1.3. Immunol. ReV. 163, 45-57. (29) van Oss, C. J. (1997) Kinetics and energetics of specific intermolecular interactions. J. Mol. Recognit. 10, 203-216. (30) Livingstone, J. R. (1996) Antibody characterization by isothermal titration calorimetry. Nature (London) 384, 491-492. (31) Tsumoto, K., Ueda, Y., Maenaka, K., Watanabe, K., Ogasahara, K., Yutani, K., and Kumagai, I. (1994) Contribution to antibody-

antigen interaction of structurally perturbed antigenic residues upon antibody binding. J. Biol. Chem. 269, 28777-28782. (32) Papanastasiou, D., Mamalaki, A., Eliopoulos, E., Poulas, K., Liolitsas, C., and Tzartos, S. J. (1999) Construction and characterization of a humanized single chain Fv antibody fragment against the main immunogenic region of the acetylcholine receptor. J. Neuroimmunol. 94, 182-195. (33) Wo¨rn, A., and Plu¨ckthun, A. (2001) Stability engineering of antibody single-chain Fv fragments. J. Mol. Biol. 305, 989-1010. (34) Todorovska, A., Roovers, R. C., Dolezal, O., Kortt, A. A., Hoogenboom, H. R., and Hudson, P. J. (2001) Design and application of diabodies, triabodies and tetrabodies for cancer targeting. J. Immunol. Methods 248, 47-66. (35) Gilkes, N. R., Jervis, E., Henrissat, B., Tekant, B., Miller, R. C., Warren, R. A., and Kilburn, D. G. (1992) The adsorption of a bacterial cellulase and its two isolated domains to crystalline cellulose. J. Biol. Chem. 267, 6743-6749. (36) Beldman, G., Voragen, A. G. J., Rombouts, F. M., Searle-van Leeuwen, M. F., and Pilnik, W. (1987) Adsorption and kinetic behavior of purified endoglucanases and exoglucanases from Trichoderma Viride. Biotechnol. Bioeng. 30, 251-257. (37) Bothwell, M. K., Daughhetee, S. D., Chaua, G. Y., Wilson, D. B., and Walker, L. P. (1997) Binding capacities for Thermomonospora fusca E3, E4 and E5, the E3 binding domain, and Trichoderma reesei CBHI on Avicel and bacterial microcrystalline cellulose. Biores. Technol. 60, 169-178. (38) Ståhlberg, J., Johansson, G., and Petterson, G. (1991) A new model for enzymatic hydrolysis of cellulose based on the two-domain structure of cellobiohydrolase-1. Bio/Technology 9, 286-290. (39) Lynd, L. R., Weimer, P. J., van Zyl, W. H., and Pretorius, I. S. (2002) Microbial cellulose utilization: fundamentals and biotechnology. Microbiol. Mol. Biol. ReV. 66, 506-577. BC060203Y