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International Immunology, Vol. 9, No. 5, pp. 771–777 © 1997 Oxford University Press Is structural flexibility of antigen-binding loops involved in the affinity maturation of anti-DNA antibodies? Satoru Miyazaki1, Junko Shimura1,2, Sachiko Hirose2, Reiko Sanokawa2, Hiromichi Tsurui2, Midori Wakiya2, Hideaki Sugawara1 and Toshikazu Shirai2 1Life Science Research Information Division, The Institute of Physical and Chemical Research (Riken), 2-1 Hirosawa, Wako, Saitama 351-01, Japan 2Department of Pathology, Juntendo University School of Medicine, 2-1-1 Bunkyo-ku, Hongo, Tokyo 113, Japan Keywords: anti-DNA antibodies, autoantibodies, autoimmunity, Ig V gene somatic mutation, Ig V region gene, molecular dynamics simulation, systemic lupus erythematosus Abstract Effects of somatic mutations in Ig variable region genes on the affinity maturation of autoantibodies were investigated using single precursor B cell-derived anti-double-stranded DNA mAb generated from an autoimmune disease-prone (NZB H NZW)F1 mouse. Analyses of DNA sequences, homology modeling on a graphic computer and molecular dynamics simulation of antigen-binding sites showed that any single site of mutation and changes in the electrostatic or hydrogen-bonding potential of the residues and in the three-dimensional structure could not solely explain the difference in DNA-binding activities. However, a significant increase in the flexibility of antigen-binding Fv loops, particularly VL CDR1 and VH CDR3, was associated with affinitymaturated anti-DNA antibodies. Such high flexibility of the Fv loops may provide the environment where the antibodies could effectively interact with antigen DNA, a model consistent with the ‘induced-fit’ hypothesis of antigen–antibody interactions. Introduction The affinity maturation of B cells involves a high rate of somatic point mutations in rearranged Ig variable (V) region genes during the process of proliferation and differentiation including class-switching (1–6). However, the correlation between structural changes in the antigen-binding site due to somatic mutations in V region genes and changes in antigen-binding activities of antibodies remain unknown. Based on sequence analyses of V genes in specific acquired immune responses to foreign antigens, somatic hypermutations were found to occur mainly in complementarity-determining regions (CDR) of V genes during the process of affinity maturation (1,3,4). In contrast, studies of our own as well as those of other workers with naturally occurring anti-DNA autoantibodies in systemic lupus erythematosus (SLE)-prone mice showed that mutations in V region genes frequently occur in both CDR and framework regions (FR) (5–11). In our preceding studies, because replacement mutations occurred at random, and differed in numbers and positions in CDR and FR, we could not correlate changes between primary nucleotide sequences and DNA-binding activities of the antibodies (5,6). Studies using X-ray crystallography have demonstrated that structures of variable and constant domains of Ig of different sources are highly conserved and that FR of these domains are essentially superimposable (12–14). Thus, the effect of point mutations in V region genes on conformational changes in Ig does not seem to be so large as previously assumed. However, as the antigen–antibody interaction is dynamic, further analyses on changes in molecular dynamics of the antigen-binding site are necessary to better understand the molecular basis of affinity maturation of antibodies. In this regard, studies using molecular dynamics simulation on a graphic computer through a conformational homology search and refinement with energy minimization procedures provided reliable models of Ig V domains (15,16). Based on this approach, we analyzed the effects of somatic hypermutations in V region genes on the affinity maturation of autoantibodies, using single precursor B cell-derived monoclonal anti-DNA Correspondence to: T. Shirai Transmitting editor: J. Berzofsky Received 26 August 1996, accepted 3 February 1997 772 Affinity maturation and Fv flexibility antibody clones with a distinct double-stranded DNA (dsDNA)-binding activity. Methods Hybridomas NZB and NZW strains of mice, originally obtained from Japan SLC (Shizuoka, Japan), were mated to produce (NZB3NZW)F1 hybrid mice. Spleen cells from a 6-month-old female mouse were fused to the non-secreting myeloma cell line P3-X63-Ag8.653, as described (5). Culture supernatants with binding activities to DNA were screened using ELISA (5). Positive clones were subsequently subcloned at least twice by limiting dilution. DNA sequencing Total cellular RNA was isolated from ~107 hybridoma cells as described in our previous study (5), and 1 µg of RNA was used for each VH and VL cDNA amplification. The first stranded cDNA was synthesized using 200 U of reverse transcriptase and 20 pmol of the C primer (for Cγ, 59-GGOCAGTGGATAGAC-39 and for Cκ, 59-GCTCACTGGATGGTGGGAAGATG39). Single-stranded cDNA was then amplified using 100 pmol each of 59 primer (for VH, 59-AGGT(C/G)(A/C)A(A/G)CTGCAG(G/C)AGTC(A/T)GG-39 or for Vκ, 59-GAAATTGTGCT (G/C)ACCCA(G/A)(T/A)CIC(C/A)A-39) and the corresponding C primer. The primers were synthesized using a Gene Assembler (Pharmacia, Uppsala, Sweden). The PCR mixtures containing 30 mM Tris–HCl (pH 8.3), 5 mM MgCl2, 0.01% gelatin, 0.2 mM of each primer, 2.5 U Tag polymerase (Takara Shuzou, Kyoto, Japan) and 0.1– 1 µg of cDNA were prepared, and amplification was carried out using a DNA thermal cycler (Perkin-Elmer-Cetus, Norwalk, CT) for 30 cycles. Each cycle consisted of denaturation at 94°C for 30 s, annealing at 55°C for 30 s and polymerase reaction at 72°C for 1 min. The length of the polymerase reaction was increased at each cycle by 15 s. PCR products of ~400 bp were isolated, using agarose gel electrophoresis, and then cloned in the pUC18 vector. Cloned and selected PCR products were sequenced using the dideoxy chain termination method and Taq dye primer cycle sequencing kits (Applied Biosystems, Foster City, CA), according to the manufacturer’s instruction. At least three independent clones of each hybridoma were sequenced to preclude misincorporated nucleotides during PCR. Measurement of DNA binding activities using DNA-coupled silica gel particles and flow cytometry To determine changes in DNA-binding activities of anti-DNA mAb, a new method using oligonucleotide-coupled silica gel particles and flow cytometry was used, as described elsewhere (6). Briefly, diol-silica, 5 µm in diameter, was activated with tresyl chloride and linked with Aminolink 2 (Applied Biosystems)-labeled oligonucleotide (59-GTGTTATAGAAATTTGATATGGAG-39). Double-stranded oligonucleotide was produced by incubating the gel particles with the complementary synthetic strand. For the measurement of DNAbinding activity of anti-DNA mAb, 53105 dsDNA-coupled silica gel particles were incubated with 30 µl of culture supernatant, with a known Ig concentration at room temper- ature for 1 h with mild shaking. After three washings with PBS (pH 7.5) supplemented with 1% BSA and 0.05% Tween 20, particles were incubated with 30 µl of appropriately diluted FITC-labeled goat anti-mouse Ig for 30 min at room temperature, washed twice and resuspended with 400 µl of PBS containing 0.2% BSA. The fluorescence intensity of DNAcoupled silica gel particles was then measured using a FACStar (Becton Dickinson, Mountain View, CA) in logarithmic amplification. The channel number showing the mean fluorescence intensity on the FACS profile was regarded as a value of the potential affinity of anti-dsDNA mAb. Model building and molecular dynamics simulation The computer graphic models of VH and VL chains of antidsDNA mAb were built, using the graphics program Insight II and Homology (Version 2.3.0, Biosym Technologies, San Diego, CA), on a Silicon Graphics (Indigo2) computer system. The general strategy for the model building was as follows: (i) selecting a reference molecule, (ii) loop searching and loop replacement, when insertion is required, (iii) checking chirality, and (iv) energy minimization (15,16). As a reference graphic model for the building of VH and VL, we used the coordinates of the reported mAb BV04-01, which is reactive with single-stranded DNA (14), because of high sequence homology to V region genes of our clonally related antidsDNA mAb. After refinement by 1000 energy minimization steps, the potential energy of all models was reduced to less than –8000 kcal/mol. Molecular dynamics of the threedimensional Fv structure models of Ig was performed for 50 ps at the temperature of 298 K and at 1 atm. The coordinates of atomic positions in the dynamics trajectory were obtained using the program Discover (Version 2.3.0, Biosym Technologies), programmed based on the report of Dauber-Osguthorpe et al. (17). To make initial loop conformations natural, the lowest energy conformations were taken. Cut-off distance for non-bonded pair interactions was set 10 Å. The extent of the protein motions seen in the simulations was assessed by calculating the fluctuations of root mean square of atomic positions in the dynamics trajectory. Results V gene mutations and DNA-binding activities in clonally related anti-DNA hybridomas We cloned 12 anti-dsDNA hybridomas (five IgM and seven IgG2b) from a single, 6-month-old (NZB3NZW)F1 mouse and sequenced V regions of heavy (VH) and light chain (VL) genes of each mAb. Among these, five IgG2b clones, BW9-7, BW9-8, BW9-9, BW9-11 and BW9-13, were thought to have originated from a single precursor, for two reasons: (i) these mAb commonly used VH J558 and JH3 as the VH region gene, and Vκ21 and Jκ2 as the VL gene, and (ii) they had identical or related junctional areas in CDR3, including potential N sequences in VH genes. Because of the lack of information on germ-line sequences of these anti-DNA clones, the nucleotide sequences of both VH and VL genes in each mAb were aligned with the consensus sequence deduced from VH and VL sequences of individual mAb to determine the site and number of somatic mutations Affinity maturation and Fv flexibility 773 Fig. 1. Nucleotide and deduced amino acid sequences for heavy (a) and light chain (b) V regions of clonally related anti-DNA mAb BW9-7, BW9-8, BW9-9, BW9-11 and BW9-13. These sequences are aligned with the consensus precursor sequence (for details, see text). Dashes indicate identity with the consensus sequence deduced from VH and VL sequences of individual mAb. The sequence of 59 end (8 bases) of the 59 primer for PCR is not shown. Amino acid sequences of CDR are boxed according to the definition of Kabat et al. (26). in the V regions. The consensus sequence may not necessarily represent the exact germline sequence. However, because members of a clone must express the same genes, any sequence differences between them are considered to be caused by a somatic mutation. As shown in Fig. 1, these five clones had different numbers of somatic mutations, with or without replacement mutations throughout CDR and FR. Thus, it was evident that somatic mutations in V region genes accumulate even after the IgM to IgG class switching of antiDNA antibodies. Comparisons of dsDNA-binding activities among mAb were made using 24mer synthetic dsDNA-coupled silica gel par- ticles as substrate and a fluorescence-activated cell sorter. The difference of DNA-binding activity was based on the difference in mean peak channels of fluorescence intensity. As shown in Fig. 2, each mAb showed a considerable difference in dsDNA-binding activity. It was of note that compared to BW9-7, four other clones, BW9-8, BW9-9, BW911 and BW9-13, showed a substantially higher dsDNA-binding activity. However, there were no common mutational positions which could account for the increased dsDNA-binding activities in higher affinity clones, compared to the activity of BW97. Using the neighbor joining method with Kimura’s distance (18), the clones, BW9-7 and BW9-9, were geneologically 774 Affinity maturation and Fv flexibility Fig. 2. Comparisons of dsDNA-binding activities between clonally related IgG2b anti-DNA mAb. dsDNA-binding activities were examined using synthetic dsDNA-coupled silica gel particles and flow cytometry, as described in Methods. Each dsDNA-coupled particle was first stained with culture supernatant of mAb at given Ig concentrations and then stained with FITC-labeled anti-mouse Ig antibodies. dsDNA-binding activities were expressed by mean channels of fluorescence intensity. suggested to be the closest pair (6) (data not shown). Nonetheless, BW9-9 showed a considerably higher dsDNAbinding activity than did BW9-7. Three-dimensional structure of antigen-binding sites of clonally related anti-DNA antibodies To determine whether changes in the three-dimensional structure of antigen-binding sites contribute to the affinity maturation of anti-DNA clones, we prepared three-dimensional structure models of the antigen-binding site consisting of Nterminal domains of VL and VH (Fv). Each Fv model was constructed by coordinating the reported VH and VL template structures of the reference anti-DNA mAb BV04-01 (14), which has a highly homologous sequence to our anti-DNA mAb, followed by refinement, using energy minimization (15,16). Figure 3 illustrates the model structure of Fv fragments of BW9-7 as a representative model structure of five clones. The attached data shows the root mean square values of deviations between the reference structure, BV04-01, and each three-dimensional model of BW9-7, BW9-8, BW9-9, BW9-11 and BW9-13, which were within the range of 2.15– 2.54 Å. In contrast, the root mean square of deviations of backbone atoms between these models of single-cell-derived anti-DNA mAb were within the range of 0.07–0.89 Å (Table 1). Thus, three-dimensional structures of antigen-binding sites, including the antigen-binding groove, were not so different and may not explain the difference in DNA-binding activities between these anti-DNA clones. Changes in flexibility of CDR loops in anti-DNA antibodies with different affinities According to the ‘induced fit’ hypothesis of the antigen– antibody interaction, it is possible that the difference in antigen-binding activity of anti-DNA mAb clones in the present Fig. 3. A representative three-dimensional structure model of anti-DNA mAb (BW9-7) refined using energy minimization. FR are displayed in trace lines and CDR in ribbons. The attached data indicates root mean square values of deviations between the reference structure (BV04-01) and each three-dimensional model (BW9-7, BW9-8, BW99, BW9-11 and BW9-13). Table 1. Root mean square of deviations (Å) of backbone atoms between models Clone BW9-7 BW9-8 BW9-9 BW9-11 BW9-8 BW9-9 BW9-11 BW9-13 0.32 0.31 0.31 0.49 0.80 0.80 0.87 0.07 0.89 0.41 study may be due to differences in their flexibility of antigenbinding loops. To investigate differences in flexibility of antigen-binding loops between low-affinity and high-affinity clones, molecular dynamics simulations were performed. The extent of protein motions seen in the simulations was assessed by calculating root mean square fluctuations of atomic positions in the molecular dynamics trajectory. Coordinates of all atoms of Fv structure models including each of three CDR loops and four FR in VH and VL were obtained at the interval of 500 fs during 50 ps molecular dynamics, and deviations of atomic positions among 100 simulated structure (snapshots of the simulation), expressed by root mean square, were Affinity maturation and Fv flexibility 775 Fig. 4. Comparison of the flexibility of Fv loops during molecular dynamics, as determined by cluster graphs. Coordinates of all atoms were obtained at the time interval of 500 fs during the simulation period of 50 ps, each set of coordinates was saved as a frame and 100 frames were stored in chronological order. The extent of protein motions seen in the simulations was estimated by calculating the root mean square fluctuations of all atomic positions including side chains among 100 frames. Results are displayed in cluster graph, in which ranges of root mean square values of 0.00–1.00 Å are shown in the black area and those of .1.00 Å are in the white area. plotted in the cluster graphs shown in Fig. 4. Deviations of atomic positions among simulated structures of 1.0 Å of root mean square value or more were regarded as a high extent of vibration (white areas in Fig. 4) and those less than 1.0 Å were regarded as a low extent of vibration (black areas). As shown by narrower black areas in Fig. 4, Fv structures of the higher affinity clones, BW9-8, BW9-9, BW9-11 and BW9-13, were more dynamic, thus more flexible than that of the lowest affinity, BW9-7. Comparisons of animated frames of all six CDR loops in VH and VL of BW9-7 showed that the VH CDR3 was the most vibration-limited loop (Fig. 5a). We then compared the extent of vibration of VH CDR3 among five clones and found that such vibration of BW9-7 was most restrained (Fig. 5). Because amino acid sequences of VH CDR3 of BW9-7 were identical to those of BW9-9, the observed limited vibration of BW9-7 VH CDR3 may be due to interaction with other loops located close to VH CDR3. Thus, we compared the distance from VH CDR3 to each VL CDR loop between BW9-7 and BW9-9. As shown in Fig. 6, while the distances between the α carbon of Lys102 in VH CDR3 and either one of the α carbons of Asn57 in VL CDR2 or that of Arg96 in VL CDR3 did not differ between the two clones (Fig. 6b and c), the distance between VH CDR3 and VL CDR1 differed between them, in which while the distance between the α carbons of Lys102 in VH CDR3 and of Asp33 in VL CDR1 of BW9-7 ranged from 10.7 to 16.1 Å, the distance between Lys102 and Gly33 of BW9-9 ranged from 8.6 to 17.0 Å (Fig. 6a). Thus, while the maximum movable distance in the low-affinity clone BW9-7 was 5.4 Å, the distance in the high-affinity BW9-9 was 8.4 Å, suggesting that VL CDR1 in the low-affinity clone BW9-7 restrains the flexibility of VH CDR3 and that certain amino acid changes in VL CDR1 are responsible for the difference in such Fv loop flexibilities between the two clones. Fig. 5. Comparison of flexibility between Fv structures of five clones was expressed by the difference in width of ribbon trace. Ribbon width corresponded to the extent of vibration in the structure during the molecular dynamics. Note that the ribbon width of VH CDR3 in the low-affinity clone BW9-7 is narrower than those in high-affinity clones. The width of this loop correlates well with the increase in DNA-binding activity. Discussion In the present study, we investigated the effects of somatic mutations in Ig V region genes on the affinity maturation of anti-DNA antibodies, using single precursor B cell-derived anti-DNA antibody clones with a distinct DNA-binding activity. The results suggested that somatic mutations in Ig V region genes affected the flexibility of antigen-binding Fv loops, and that there was correlation between an increase in flexibility of Fv loops and affinity maturation of anti-DNA antibodies. It has been reported that a common and characteristic feature of anti-DNA mAb from SLE-prone mice is the appearance of the cationic amino acid residue Arg in VH (7–11), and the process of both selection and somatic mutation may be involved in the enrichment of Arg in VH CDR3 (8). Arg is the most versatile amino acid for DNA binding, in that it can form ionic bonds with the negatively charged phosphodiester backbone of DNA and also donate up to five hydrogen bonds. Thus, it is suggested that the increase in the number of Arg in the antigen-binding site contributes to the increase in affinity of antibodies to DNA. This is indeed the case with the highest affinity clone BW9-11, which had three repeated Arg in the junction of VH FR3 and CDR3, in contrast to two Arg in the same region in the lowest affinity clone BW9-7 (Fig. 1). However, such was not necessarily the case for other higher affinity clones BW9-8, BW9-9 and BW9-13, i.e. these three clones had no additional substitutions to Arg in CDR, compared to the lowest affinity clone, BW9-7. In addition to Arg, Asn and Lys also contribute to DNA binding (10). Compared to the presence of two Asn in CDR of BW9-7, higher affinity clones BW9-8, BW9-11 and BW9-13 had three, four and five Asn respectively. However, another high-affinity clone BW9-9 had only one Asn. As for Lys, the 776 Affinity maturation and Fv flexibility Fig. 6. Comparison of the transition of distance between VH CDR3 and VL CDR loops over the period of molecular dynamics. The cationic amino acid residue located in each loop was selected as an effective point for the antigen-binding site and the distance between α carbons of the selected amino acids was measured within the trajectory and plotted against the frame. The selected positions of amino acids on each loop are as follows: VL CDR2, Lys57; VL CDR3, Arg96; VH CDR3, Lys102. Since the VL CDR1 lacks cationic residues at the top portion of the loop, Asp33 or Gly33 was taken to measure the distances. (a) Comparison of distance between VL CDR1 and VH CDR3. (b) Comparison of distance between VL CDR2 and VH CDR3. (c) Comparison of distance between VL CDR3 and VH CDR3. Solid line indicates the results of BW9-7 and dotted line BW9-9. number in CDR of low-affinity clone BW9-7 was the largest of all the five clones (Fig. 1). Substitutions of certain amino acids to negatively charged Glu and/or Asp may be related to the low DNA-binding activity. The lowest affinity clone BW9-7 contained Asp33 instead of Gly33 in VL CDR1. However, the higher affinity clones BW9-8, BW9-9 and BW9-13 had an additional Glu56 in VH CDR2, Glu27 in VL CDR1 and Asp56 in VH CDR2 respectively. Taken collectively, the amino acid substitutions to Arg, Asn, Lys, Glu and Asp in CDR loops do not simply explain the difference in DNA-binding activities of anti-dsDNA antibodies in the present study. Both the lock-and-key (19) and induced fit (20–25) type models have been used to describe antibody–antigen recognition. Wilson et al. (21) analyzed the shape of the antigenbinding site, based on studies of a number of Fab complexes with antigens ranging from small haptens to proteins. Although total surface areas buried by various antigens were often similar, the surface contours seen by the antigen differed and the difference in the two conformational forms of the Fab (liganded and unliganded) illustrated the induced fit of an antibody to an antigen (20–25). Based on these hypotheses, we examined the relationship between flexibilities of antigenbinding Fv loops, as determined using molecular dynamics simulations and DNA-binding activities of clonally related anti- DNA mAb. Our findings were: (i) Fv structures of higher affinity clones were more dynamic, thus more flexible than the structure of the lowest affinity clone BW9-7, and (ii) the flexibility of VH CDR3 and VL CDR1 seems to correspond to the affinity change. Molecular dynamics simulations in the present studies were carried out in vacuo. Further studies using simulations in the presence of a 5 Å layer of water are ongoing in our laboratories and our preliminary results are in principle consistent with the observations in vacuo. As the low-affinity BW9-7 and the high-affinity BW9-9 originated from a single precursor B cell, and as amino acid sequences of VH CDR3 of BW9-7 and BW9-9 were identical, certain somatic replacement mutations which occurred in VL CDR1 must be responsible for the observed difference in the loop flexibility. There are three amino acid changes in VL CDR1 between BW9-7 and BW9-9, one Lys27 versus Glu27, one Asp33 versus Gly33 and one Asn35 versus Ser35 (Fig. 1). There are at least two plausible interpretations for the observed difference in flexibilities of VL CDR1 and VH CDR3 between BW9-7 and BW9-9. First, changes in the loop flexibility may be caused by the occurrence of certain amino acids. The amino acid substitution to Gly may increase the loop flexibility, because Gly residues have more conformational freedom than any other amino acids. The side chain of Asn frequently hydrogen bonds to the main chain, thereby stabilizing the local conformation. The substitutions from Gly33 and Ser35 in BW9-9 to Asp33 and Asn35 in BW9-7 may decrease the flexibility of VL CDR1 in BW9-7. Such restricted flexibility of VL CDR1 in BW9-7 may also be related to the observed decrease in flexibility of VH CDR3. Alternatively, the electrostatic interaction between the negatively charged Asp33 in VL CDR1 and the cationic residue Lys102 in VH CDR3 in BW9-7, which is located close to Asp33 in VL CDR1, may form the basis of the restricted flexibility of VL CDR1 and VH CDR3. In BW9-9, this restriction may be released because of Asp33 to Gly33 substitution in VL CDR1. Although there is additional substitution from Lys27 to negatively charged Glu27 in VL CDR1 of BW9-9, the site is not close enough to interact with positively charged Lys102 in VH CDR3. Taken collectively, our results suggest that in addition to the reported importance of cationic amino acid residues in Fv loops, the loop flexibilities may also influence the DNAbinding reaction of antibodies. The high flexibility of VH CDR3 and VL CDR1 loops may provide the environment where electrophilic amino acid residues along the antigen-binding groove of antibody could effectively interact with antigen. 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