Study of B72.3 combining sites by molecular modeling and site-directed mutagenesis

Jim Xiang1,2, Maheswaran Srivamadan4, Raju Rajala3 and Zongchao Jia4

1 Departments of Microbiology, Oncology 3 Pathology, Saskatoon Cancer Center, University of Saskatchewan, Saskatoon, Saskatchewan S7N 0W0 4 Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
A B72.3 Fab/sTn2 complex was modeled from the known structure of B72.3 Fab and the dimeric Tn-serine cluster (sTn2). In the complex model, the side chains of 15 heavy- and light-chain complementarity-determining region (CDR) residues and the main chains of two light-chain CDR residues contact the sTn2 epitope. Among 15 CDR residues which contact sTn2 in the model, two heavy-chain residues (Ser95 and Tyr97) and light-chain CDR residue (Tyr96) have been confirmed in a previous study. To test the accuracy of the computational model, further site-directed mutagenesis was performed by alanine scanning on the remaining 12 residues that are predicted in the model to have side-chain interactions with sTn2. Of these 12 mutants, eight that are all from the heavy-chain (His32Ala, Ala33Leu, Tyr50Ala, Ser52Ala, Asn52Ala, Asp56Ala, Lys58Ala and Tyr96Ala) had significantly reduced sTn2 affinities, and four consisting of three light-chain mutations (Asn32Ala, Trp92Ala and Thr94Ala) and one heavy-chain mutation (His35Ala) retained wild-type sTn2 affinity. On the whole, this evidence suggests that the complex model, although not perfect, is correct in many of its features. In a more general vein, these results lend credibility to the computational modeling approach for the study of the molecular basis of antigen–antibody complexes.

Keywords: binding affinity/CDR residues/molecular modeling/site-directed mutagenesis


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Immunoglobulin G molecules consist of pairs of associated heavy (H) and light (L) polypeptide chains, each of which contains multiple globular domains. All of these domains exhibit a similar folding pattern, often described as a sandwich of three and four stranded antiparallel ß-sheets that are linked by disulfide bonds. Sequence comparisons between H- and L-chain variable (V) domains reveal that each domain has three complementarity-determining regions (CDRs) flanked by four framework regions (FRs) of less variable sequence (Kabat et al., 1991Go). Antibody combining sites are formed by the juxtaposition of six CDRs appearing as loops at one end of a ß-sheet barrel, three loops from VH and another three from VL. These CDR loops form a surface that interacts with an antigen. Note that only a subset of the presented CDR residues makes contact with an antigen (Wilson and Stanfield, 1993Go). Not surprisingly, amino acid substitutions at these contact CDR residues can significantly alter both the antibody specificity (Kussie et al., 1994Go) and the antigen-binding affinity (Allen et al., 1988Go; Sharon, 1990Go).

While carbohydrate recognition by proteins is certainly a subject replete with intrinsic interest (Lemieux, 1989Go), it is the practical implications of this molecular recognition process that are fueling the current interest in antibody engineering. The realization of the considerable potential of antibody engineering, however, requires a deeper understanding of the intermolecular interactions that govern the affinity and specificity involved in antigen binding. Towards this end, crystallographic analysis of antigen–antibody complexes has become a major appproach in the study of the molecular basis of antibody combining sites by visualization of CDR loop conformation and identification of CDR contact residues. Crystallographic studies of several carbohydrate–antibody complexes have been reported, providing important insights into the molecular basis of combining sites of these antibodies (Cygler et al., 1991Go; Bundle et al., 1994Go; Zdanov et al., 1994Go). Unfortunately, crystallographic studies are major undertakings without guarantees of success. The difficulties involved in obtaining sufficiently pure protein, screening conditions for crystallization and complex formation and in final structure determination are not unsubstantial. Because of these difficulties and the recent advances in affordable computational power, an alternative to crystallography is the use of molecular modeling in which an computational model of antigen–antibody complex is generated (Ruff-Jamison and Glenney, 1993Go). Although this approach produces only theoretical structures and thus lacks empirical foundations, it has the potential to be used synergistically with other biochemical techniques, both directing other studies, and being enhanced by the outcomes of such studies.

B72.3 is a mouse monoclonal antibody with specificity for the human tumor-associated glycoprotein TAG72 (Thor et al., 1986Go). The TAG72 epitope recognized by the B72.3 antibody is an O-linked carbohydrate (NeuAc2-6{alpha}GalNAc{alpha}1-O-Ser/Thr) (sTn) (Kjeldsen et al., 1988Go). Recent experiments have demonstrated that the minimal epitope for the B72.3 antibody is the dimeric sTn-serine cluster (sTn2) (Reddish et al., 1997Go). The B72.3 antibody has been extensively employed for immunodetection of human malignancies, revealing a 70–80% positive rate including 20% occult lesions (Carrasquillo et al., 1988Go; Percivale et al., 1996). However, the molecular basis of the recognition by B72.3 combining sites is not well understood. Structural details would, of course, improve our knowledge of the molecular basis of antigen recognition by B72.3. In an analogous situation, the X-ray structure of an anti-tumor antibody in complex with carbohydrate antigen provided a rationale for mutagenesis experiments that have resulted in CDR loop mutants with increased affinity for nLev antigen and tumor cells (Jeffrey et al., 1995Go). The crystal structure for the B72.3 Fab' revealing some important structural features of B72.3 combining sites has been determining and refined to a resolution of 3.1 Å with an R-factor of 17.6% (Brady et al., 1991Go). However, at present, no structural data on the B72.3/TAG72 complex are available.

In this study, we developed a computational model of B72.3/sTn2 complex and identified 17 contact CDR residues based upon this complex model. The modeling study helped us to plan a mutagenesis strategy for conducting site-directed mutagenesis at predicted contact CDR residues. The binding affinities of these mutant antibodies were measured in a solid-phase radioimmunoassay (RIA) and compared with that of the original antibody cB72.3 (Xiang et al., 1990Go).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Cell lines and antigen

The SP2/0Ag14 melanoma cell lines that lack the expression of internal H- and L-chains of myeloma protein were purchased from the American Type Culture Collection (ATCC, Rockville, MD). The transfectoma cell line (Xiang et al., 1990Go), which secretes a mouse/human chimeric anti-TAG72 antibody cB72.3, was maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) and geneticin (0.5 mg/ml). Bovine submaxillary gland mucin (BSM) (Sigma Chemical, St. Louis, MO), which contains the TAG72 epitope (Xiang et al., 1990Go), was used as the antigen source in this study.

Molecular modeling

The previously determined structure of the B72.3 Fab' fragment (Brady et al., 1992Go) was used as a template. The 3-D structure of the single sTn epitope was constructed using a number of relevant fragment structures obtained from the Cambridge Structure Database (University of Cambridge, UK). These include serine, sialic acid and galactosamine. Essentially, a single sTn was built from two galactosamine moieties via a 2–6 alpha connection, with serine and sialic acid linked at the two ends. In the light of the U-shaped binding channel of the epitope (see Results and Discussion), a preliminary model of the double sTn molecule was then readily constructed by joining two single sTn moieties together which would be further optimized in later energy minimization calculations. It was known from our earlier site-directed mutagenesis experiments that residues Asp55, Ser95 and Tyr97 in the H-chain and Tyr96 in the L-chain appeared to affect sTn epitope binding (Xiang et al., 1996Go). Taking this into consideration, the initial docking position of a single sTn epitope with respect to the B72.3 Fab was determined using the program DOCK (Kuntz, 1992Go). The epitope was treated as a rigid body in the first round of docking, but all atoms were allowed to move individually in subsequent rounds of optimization. Following the positioning of the single sTn, the double sTn (sTn2) epitope was manually placed in the binding pocket of B72.3 Fab. The complex model was subjected to Powell energy minimization implemented in X-PLOR (Brünger, 1992Go), where 2000 cycles of energy minimization was carried out and energy converged. The parameter file used in the energy minimization was compiled and modified using the similar parameter files for serine, sialic acid and glucosamine fragments, available at the website http://xplor.csb.yale.edu/hetero. Note that glucosamine differs from galactosamine in only the orientation of a single hydroxyl group and the parameter file was readily converted. All graphics analysis was carried out on SGI graphics computers.

Site-directed mutagenesis

Amino acid sequences of the previously cloned cB72.3 VH and VK genes (Xiang et al., 1990Go, 1992Go) are numbered sequentially and according to Kabat's method (Kabat et al., 1991Go) (Figure 1Go). CDR loops L1, L2 and L3 adopt a canonical structure of types 2, 1 and 1, while CDR loops H1 and H2 belong to types 1 and 2, respectively (Chothia et al., 1989Go). As observed from the modeled B72.3/sTn2 complex, 17 CDR contact residues are thought to have hydrogen bonds and van der Waals interactions with the sTn2 epitope, including 11 H-chain CDR residues (His32, Ala33, His35, Tyr50, Ser52, Asn54, Asp56, Lys58, Ser95, Tyr96 and Tyr97) and six L-chain CDR residues (Asn32, Phe91, Trp92, Gly93, Thr94 and Tyr96). Among them, epitope interactions with two H-chain contact CDR residues (Ser95 and Tyr97) and one L-chain contact CDR residue (Tyr96) have been previously demonstrated by site-directed mutagenesis (Xiang et al., 1996Go). Of the remaining 14 residues found to interact with sTn2 in our model, two L-chain CDR residues (Phe91 and Gly93) have interactions with the sTn2 epitope through their main-chain atoms. It is possible that mutations of these two residues might introduce some steric effects that may interfere with main-chain interactions, although it is expected to be unlikely since the side chains in question point away from the sTn2 epitope. Consequently, these sites were not selected for alteration by mutation, thus avoiding any compliction in our results. To examine whether the remaining 12 CDR residues influence the binding affinity for the TAG72 antigen, these CDR residues were individually changed by site-directed mutagenesis either to eliminate hydrogen bonds or to alter van der Waals interactions at these positions. Twelve oligonucleotides (oligo 1–12) complementary to different areas of VH and VK regions of the cB72.3 (Xiang et al., 1990Go, 1992Go) were designed and synthesized for amino acid substitutions. These 12 oligonucleotides includes oligo 1 (5'cttca ctgac GCtgc tattc ac 3'), oligo 2 (5' cactg accat CTtat tcact gg 3'), oligo 3 (5'ccatg ctatt GCctg ggcga ag 3'), oligo 4 (5'atgga ttgga GCtat ttctc cc3'), oligo 5 (5'tggat atatt Gctcc cggaa at 3'), oligo 6 (5'ttctc ccgga GCtga tgata tt 3'), oligo 7 (5'tgatg atatt GCgta caatg ag 3'), oligo 8 (5'taaaa gatcg GCcta cggcc ac 3'), oligo 9 (5'tgatg atatt GCgta caatg ag 3'), oligo 10 (5'tattt acagt GCttt agcat gg 3'), oligo 11 (5'tcaac atttt GCggg tactc cg 3') and oligo 12 (5'tttt gggt Gctcc gtaca gt 3'). Note the mutated nucleotides are capitalized. These mutations include nine H-chain mutations (His32Ala, Ala33Leu, His35Ala, Tyr50Ala, Ser52Ala, Asn54Ala, Asp56Ala, Lys58Ala and Tyr96Ala) and three L-chain mutations (Asn32Ala, Trp92Ala and Thr94Ala). Mutations were introduced into M13mp1-VH or M13mp19-VK by these 12 oligonucleotides through site-directed mutagenesis (Zoller and Smith, 1983Go) to form 12 plasmids (M13mp19-VHm1–9 and M13mp19-VKm1–3), each containing a mutant B72.3 VH or VK region of cDNA fragment with the RNA splice junction consensus sequence (5'caggtagt 3') at its 3' end (Xiang et al., 1990Go, 1992Go). Sequences of these mutant VHm1–9 and VKm1–3 regions were verified by the dideoxy nucleotide sequencing method (Sanger et al., 1977Go).



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Fig. 1. Amino acid sequences of the cB72.3–1–3 (A) VH and (B) VK regions. The one-letter amino acid code is used. Amino acids are numbered (i) sequentially and (ii) according to Kabat's method. Demarcated are respective framework regions (FR), complementary-determining regions (CDR) and joining segment (J).

 
Construction of expression vectors

The mutant VHm1–9 cDNA fragments (EcoRI/SalI) were purified from M13mp19-VHm1–9 by EcoRI/SalI digestion and introduced into an EcoRI/XhoI site in the multiple cloning region of mpSV2neo-EP1-C{gamma}1 (Xiang et al., 1990Go) to form the chimeric H-chain expression vectors mpSV2neo-EP1-VHm1–9. The mutant VKm1–3 cDNA fragments (SalI) were purified from the M13mp19-VKm1–3 by SalI digestion and introduced into a SalI site in the multiple cloning region of mpSV2gpt-EP1-CK (Xiang et al., 1992Go) to form the chimeric L-chain expression vector mpSV2gpt-EP1-VKm1–3-CK. The ligated chimeric H- or L-chain expression vectors containind either the neo gene for geneticin selection or the gpt gene for mycophenolic acid selection, and a complete transcription unit including enhancer (E), immunoglobulin promoter (P1), mutant VHm1–9 or VKm1–3 region cDNA fragments and human genomic {gamma}1 (C{gamma}1) or K constant region (CK). These 12 expression vectors mpSV2neo-EP1-VHm1–9C{gamma}1 and mpSV2gpt-EP1-VKm1–3CK were used for the expression of 12 mutant chimeric cB72.3m1–12 antibodies (Xiang et al., 1990Go, 1992Go).

Expression of mutant chimeric antibodies

The chimeric H- and L-chain expression vector DNAs of mpSV2neo-EP1-VHm1–9C{gamma}1 and mpSV2gpt-EP1-VKm1–3CK were transfected into the SP2/0Ag14 cells by electroporation as described previously (Xiang et al., 1990Go, 1992Go). Cells were selected for growth in media containing either geneticin at 2.0 mg/ml or mycophenolic acid at 25 µg/ml. After 14 days, growth supernatants were screened in human {gamma}- and K-chain capture ELISA for examining the expression of chimeric cB72.3m1–9 antibodies and the mutant chimeric L-chains (Km1–3) respectively (Xiang et al., 1990Go, 1992Go). The positive clones derived from the SP2/0Ag14 cell line for the expression of mutant chimeric L-chains were further transfected with the chimeric H-chain expression vector DNA mpSV2neo-EP1-VHC{gamma}1 (Xiang et al., 1990Go) and selected for growth in media containing both geneticin (2 mg/ml) and mycophenolic acid (25 µg/ml). The growth supernatants were then screened in human {gamma}-chain capture ELISA for examining the expression of mutant chimeric antibodies cB72.3m10–12. Mutant chimeric antibodies cB72.3m1–12 were further purified from their supernatants by Protein A-Sepharose column chromatography (Xiang et al., 1990Go). Protein concentrations were determined using a Bio-Rad protein assay kit (Bio-Rad, Sunnyvale, CA) according to the manufacturer's instructions.

Affinity constants

The affinity constants of the original cB72.3 antibody and the mutant cB72.3m1–12 antibodies were determined in a solid-phase RIA as described previously (Xiang et al., 1996Go). Briefly, serial dilutions of mutant antibodies were added to each mucin-coated well (in triplicate) of the first microtiter plate for incubation overnight at 4°C. The supernatants of each well, which contain the free mutant antobodies, were transferred to each well of the second microtiter plate that had previously been coated with goat anti-human IgG antibody. The amounts of bound and free antibodies in the first and the second plates were measured by using the 125I-labeled goat anti-human IgC antibody. Scatchard plot analysis was carried out to calculate the affinity constants (Ka) (Scatchard, 1949Go). The ratios of the concentration of bound to free antibody were plotted against the concentration of bound antibody. The slope represents Ka of each mutant antibody.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Modeling of the B72.3/TAG72 complex

The single sTn complex model is unable to satisfy all experimentally characterized interactions involving residues Ser95 and Tyr97 in the H-chain and Tyr96 in the L-chain (Xiang et al., 1996Go). Some, but not all, of the experimentally confirmed contact residues are able to interact simultaneously with the single sTn epitope. However, the single sTn model provides an important clue as to how the shape complementarity between antibody and antigen can be maximized by the judicious placement of residues known to interact with the antigen. The binding cavity resembles a U-shaped channel centered near Tyr96. The size and overall shape of the binding cavity suggest that sTn2, not sTn, may in fact be the preferred epitope. Our subsequent modeling studies indicate that the sTn2 epitope possesses a high degree of structural and chemical complementarity with the binding site. Also, the interactions observed in such a complex are consistent with all of the experimental mutagenesis data obtained previously (Xiang et al., 1996Go). The model is also supported by some recently reported experimental evidence (Reddish et al., 1997Go), in which the binding of B72.3 antibody to the TAG72 antigen has been shown to be inhibited by the sTn2 epitope, but not by sTn. Based on the sTn2 complex model, we analyzed various interactions between the B72.3 Fab and the sTn2 epitope. In this model, numerous polar atoms are at appropriate geometries to form hydrogen bonds, and contact distances between B72.3 Fab and sTn2 suggest that many less specific van der Waals interactions should form. Table IGo summarizes all potential interactions between the B72.3 antibody and the sTn2 epitope, as identified from the complex model. As seen from Table IGo, a total of 17 CDR contact residues are predicted to interact with the sTn2 epitope by way of either hydrogen bonds or van der Waals interactions. These include nine H-chain CDR residues (His32, His35, Tyr50, Ser52, Asn54, Asp56, Ser95, Tyr96 and Tyr97) and four L-chain CDR residues (Asn32, Phe91, Gly93 and Tyr96) having both hydrogen bonds and van der Waals interactions with the sTn2 ligand, and two H-chain CDR residues (Ala33 and Lys58) and two L-chain CDR residues (Trp92 and Thr94) having only van der Waals interactions with the sTn2 epitope. The overall ribbon illustration of the complex, a close-up view of the binding site and a stereo diagram showing interations are given in Figures 2Go, 3 and 4GoGo, respectively.


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Table I. Contact CDR residues identified in the sTn2 complex modela
 


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Fig. 2. Overall backbone illustration of the double sTn complex. Secondary structure of Fab is highlighted. The antigen is shown in ball-and-stick model. This diagram was prepared using Molscript (Kraulis, 1991Go).

 


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Fig. 3. Close-up view of the binding site. Fab is presented as electrostatic surface. The epitope is shown in stick model. Contact CDR residues are illustrated (last letter following the residue number, H or L, stands for heavy- or light-chain). This diagram was prepared using Grasp (Nicholls et al., 1993Go).

 


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Fig. 4. Stereoview of sTn2 binding and interaction. Residue labeling as in Figure 3Go. Hydrogen bonds are highlighted by dashed lines and distances (in Å). This diagram was prepared using Molscript (Kraulis, 1991Go).

 
Site-directed mutagenesis and affinity constant of mutant antibodies

Among these 17 contact CDR residues, two H-chain CDR residues (Ser95 and Tyr97) and one L-chain CDR residue (Tyr96) have been previously demonstrated by site-directed mutagenesis to be involved in epitope contact (Xiang et al., 1996Go). Amino acid substitution at these positions resulted in a significantly decreased affinity towards the TAG72 antigen. In addition, our previous studies have also shown that some atomic interactions that form between CDR and FR residues may play an important role in influencing TAG72 binding by altering CDR loop conformations (Xiang et al., 1995Go, 1999Go).

Site-directed mutagenesis was performed at 12 positions, nine in H-chain (His32, Ala33, His35, Tyr50, Ser52, Asn54, Asp56, Lys58 and Tyr96) and three in L-chain (Asn32, Trp92 and Thr94). All positions except for Ala33 were subjected to mutagenesis by alanine scanning, which is a useful tool for the identification of functionally important side chains (Jin and Wells, 1994Go). Because the short side chain of Ala33 has van der Waals interactions with the sTn2 epitope, a mutation of Ala33 to Leu, a residue with a larger side chain, was designed to generate steric clash with the epitope. Site-directed mutagenesis resulted in the generation of 12 mutant chimeric antibodies cB72.3m1–12. The affinity constants of these 12 mutant antibodies for the TAG72 antigen were determined in a solid-phase RIA. As shown in Table 2,Go the affinity reduction of mutant antibodies, which ranged from 8- to 84-fold compared with that of the original cB72.3 antibody, was found in eight mutant antibodies (cB72.3m1–2 and cB72.3m4–9). Four mutant antibodies (cB72.3m3 and cB72.3m10–12) had the same affinity constant for the TAG72 antigen as the original cB72.3 antibody.


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Table II. Comparison of affinity constants of mutant antibodiesa
 
Correlation between model prediction and experimental results

The sTn2 complex, modeled using limited knowledge of contact residues at the initial stage of this study, predicted that 12 additional residues interact with the sTn2 epitope through their side chains. Our later mutagenesis experiments confirmed this assumption that most of these predictions are correct with only four exceptions including one H-chain mutation of His35Ala and three L-chain mutations of Asn32Ala, Trp92Ala and Thr94Ala. The majority of these predicted residues have hydrogen bonding (Figure 4Go) and/or van der Waals interactions with the sTn2 ligand. In cases where the amino acid was changed to Ala, the shortened side-chain would be unable to interact with the antigen, causing a decrease in binding affinity. In the case of Ala33, it was predicted that van der Waals interactions could occur between the antigen and the side chain of Ala33; any bulkier replacement should cause steric clash. Upon changing to Leu, the binding affinity of the epitope did indeed decrease drastically (84-fold), supporting our model's prediction. Of all of the mutants, Tyr96, is a particularly interesting residue because it is strategically positioned in the middle of the `U'-shaped sTn2 binding channel (Figure 3Go). Binding was weaked by the Tyr->Ala mutation by 40-fold, suggesting that Tyr 96 plays a critical role in antigen binding, perhaps because its central location causes it to make many contacts with the antigen. Taken together with the previously reported Tyr96Phe mutation (Xiang et al., 1996Go), which had no effect on sTn2 binding, it seems to suggest that the hydroxyl group of Tyr96 is probably not as important as van der Waals interactions derived from the aromatic moiety.

As mentioned, four residues predicted to decrease sTn2 affinity retained wild-type affinity. The two L-chain residues Asn32 and Trp92 were predicted to contact one `end' of the `U'-shaped sTn2 (upper left area in Figure 3Go). We envisage that the terminal portion of sTn2 could be rather flexible and may not in fact form much firm contact with the B72.3. Even in the static model, Trp92 is not very close to sTn2. Of the 10 atoms in the Trp92 side chain, only two make van der Waals interactions with sTn2. Thus, the absence of any activity loss in these mutants is not wholly incompatible with our model. Similarly, the mutation at L-chain residue Thr94, which also had no effect on activity, probably reflects the fact that there are no hydrogen bonds and only weak van der Waals interactions between this residue and the sTn2 epitope. Surprisingly, however, is the fact that the H-chain mutation His35Ala did not affect the affinity of mutant antibody cB72.3m3 for the TAG72 antigen. This has been difficult to rationalize since His35 would be involved in both H-bonding and van der Waals interaction. It may be a reflection of the limitations of the modeling and its accuracy in predicting contact residues. Interestingly, in our previous experiments, we have shown that the mutation of His35Leu did not affect the TAG72 binding (Xiang et al., 1996Go). The inconsistency, and perhaps some of the others mentioned above, might also be explained by the concept of `functional epitope of the antibody', in which only some of the contact residues observed by X-ray crystallography are found to dominate the energetics of antigen binding (Davies et al., 1990Go; Kelley and O'Connell, 1993).

Our previous study showed that a single amino acid substitution at three H-chain CDR residues (Asp55, Ser95 and Tyr97) and at one L-chain CDR residue (Tyr96) resulted in affinity reductions of 20-, 8-, 15- and 40-fold, respectively (Xiang et al., 1996Go). These results are consistent with what is predicted from our complex model. Three of these four residues are found in contact with sTn2 in our computational model. The fourth residue in this group, Asp55, while not in direct contact with the sTn2 epitope, does nevertheless have potential to affect epitope binding. It can form a strong salt bridge with H-chain FR residue Lys74, which plays a major role in stabilizing part of the H-chain CDR2 loop (Ser52–Pro52a–Gly53–Asn54–Asp55–Asp56). This loop contains several contact CDR residues, including Ser52, Asn54 and Asp56.

In summary, we have developed a plausible B72.3/sTn2 complex based on the limited experimental results obtained previously. This model assisted in the planning of a series of point mutations. The decreased activities found for resulting mutants, in turn, lend credibility to our complex model. The qualitative correlation of modeling analysis with the experimental results is satisfactory. Identification of the important contact residues is very important in understanding the B72.3 combining sites and the critical interactions between antibody and antigen. The results of this study also suggest that, in the absence of a complex structure, computational modeling is a useful approach for the study of the molecular basis of antigen–antibody complexes.


    Notes
 
2 To whom correspondence should be addressed Back


    Acknowledgments
 
This study was supported by research grants from the Saskatchewan Cancer Agency (J.X.) and the Medical Research Council of Canada (Z.J.). The technical support of Yuahua Sha and Yimin Qi in this study is appreciated. We thank Brent Wathen for his help with the manuscript.


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 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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Received August 10, 1999; revised December 16, 1999; accepted February 20, 2000.





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