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Key Words: immunoglobulin • somatic mutation • X-ray crystallography • antibody affinity • affinity maturation
The antibody response to phosphocholine (PC) is biologically interesting because of the frequent expression of PC on pathogenic bacteria and nematodes where the haptenic epitope is coupled to a variety of carrier structures, including carbohydrates and proteins 91011. The murine response to PC–protein is a well-characterized model system used to study the shift in specificity to include the carrier as the immune response progresses 712131415. After immunization with PC coupled to KLH via a diazophenyl linker, the antibody population specific for PC at the onset of the primary response (group I antibodies) shifts to one that includes the carrier as part of a more complex epitope. Thus the response matures, evolving toward a population that has a strong requirement for PC in the context of the nitrophenyl linker (group II antibodies). The emergent group II antibody population requires recruitment and expansion of B cell clones expressing novel V gene combinations rarely seen in the primary response 7. The expansion and eventual dominance by a minor component of the initial antibody repertoire points to the importance of carrier determinants in selecting and stimulating lymphocytes of the group II phenotype that predominate in the memory pool.
We have shown previously that somatic mutation in a prototype group II hybridoma, M3C65, results in a dramatic increase in affinity for antigen. High affinity for p-nitrophenyl phosphocholine (NPPC), a hapten that mimics the diazophenyl linkage between PC and protein, is attributable to mutations at three positions in CDR2 of the M3C65
In this study, we use crystallographic and affinity analysis to reveal the molecular basis for the repertoire shift that occurs after immunization with PC–KLH. We determine the intrinsic affinity of M3C65 in its mutated and germline forms for compounds representing various portions of the carrier protein coupled to PC. We also solved the crystal structure of the single-chain Fv (sFv) of this antibody complexed to NPPC. The M3C65 combining site has an unusual requirement for direct interaction of the LCDR2 with NPPC, a finding that is rare for binding of small hapten molecules 18. The structure of the complex provides a stereochemical basis for emergence of group II dominance in a secondary response, and allows evaluation of the direct and indirect effects of L chain modification by somatic mutation in generating high affinity in the M3C65 hybridoma.
Transfectant Antibodies and Ka Determination.
Crystallization of M3C65 sFv Complexed to NPPC.
Data Collection and Structure Solution.
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Affinity maturation of serum antibodies occurs during the immune response to T cell–dependent antigens 1 and can be critical for protective immunity. In many antibody responses, much of the diversity exhibited by secondary and memory antibody populations is provided by somatic mutation of primary gene combinations 2345. However, another level of diversity can also be provided by recruitment of new germline VH and VL genes during the response, termed repertoire shift 6. These new gene combinations are frequently modified by mutation, providing high affinity for the immunogen 4678. The molecular basis of this selective process is not entirely clear, but may depend in part on a shift in specificity toward recognition of the hapten plus carrier determinants. 
1 L chain 16. Accumulation of replacement mutations at these key positions in the L chain occurs repeatedly in B cells undergoing affinity maturation in response to PC–KLH 17.
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Synthesis and Expression of M3C65 sFv.
cDNA copies of the VH and VL genes of the hybridoma M3C65 16 were synthesized from mRNA using random hexamer primers (Amersham Pharmacia Biotech). PCR amplification was carried out using a primer for the 5' portion of V1 (5'agatcgcatgccatggGACAGGCTGTTGTGACTCATGGAA-3') and a primer for the 3' portion of J1 plus the 5' part of the (Gly4Ser)3 linker (5'-cggacccaccaccgcccgagccaccgccaccACCTAGGACAGTCAGTTTGGT-3'). Amplification of the VH M141/D/JH3 gene was carried out using a primer for the 3' part of the linker plus the 5'VH (5'-tcgggcggtggtgggtccggaggcggatctCAGGTGCAGCTGAAGGAGTCAG-3') and a primer for the 3' end of JH3 plus two stop codons and an NcoI site (5'-agatcgcatgccatggttatcaTGCAGAGACAGTGACCAGAGTCCC-3'). NcoI sites are underlined, and V region coding sequences are capitalized. The VL and VH genes were joined by PCR with the linker (Gly4Ser)3 in the order VL-linker-VH. The amplified sFv DNA was ligated into the NcoI site of the pET3d vector (Novagen), sequenced, and transformed into Escherichia coli Bl21(DE3). DNA sequencing of clone 2-1.2 showed that the deduced protein sequence was identical to the M3C65 hybridoma. The induced protein sFv inclusions (24.9 kD) were denatured and refolded as described 19. Renatured sFv fragments were purified by affinity chromatography on PC–sepharose as described for intact antibody 12.
Antibodies expressing mutations at L chain positions 52, 53, or 55 produced by site-directed mutagenesis and the method for determination of binding constants by fluorescence quenching have been described 16. Stable transfectants cotransfected with the M3C65 H chain and the various mutant L chain constructs were made in the SP2/0 cell line and purified as described previously 16. The synthesis and structure of monoconjugates of PC coupled to tyrosine, histidine, and the Gly-Tyr-Ala tripeptide have been described; PC is coupled to Tyr on the tripeptide 20. These compounds were provided by Drs. D. Peyton, E. Barbar, and H. Moulton (Portland State University, Portland, OR). A 15–amino acid peptide, acetylated at the NH2 terminus (Peptide Express), was designed to contain one haptenation site and to have 
-helical secondary structure 21. The peptide, Ser-Asp-Ala-Leu-Ala-Glu-Met-Tyr-Glu-Leu-Met-Ala-Val-Asp-Gly, was coupled to PC at the Tyr residue as described previously 22. PC–histone was coupled by the same method and has a PC/protein ratio of 2:1.
The purified sFv at 400 µg/ml was dialyzed against 20 mM Tris, pH 7.6, concentrated to 5.6 mg/ml, and NPPC was added to a final concentration of 2 mM. Crystals were grown at room temperature by vapor diffusion from a reservoir buffer that contained 1.5 M NaH2PO4/K2HPO4, pH 6.5, and 0.1 M Hepes buffer (GIBCO BRL). The crystals are orthorhombic, take the space group P212121 (a = 130.5 Å, b = 35.9 Å, c = 50.5 Å), and contain one monomer in the asymmetric unit.
X-ray intensity data were collected at room temperature using a RAXIS IV (Rigaku) imaging plate system and a Rigaku RU300 rotating anode X-ray generator equipped with double-focusing mirrors and operating at 50 kV and 100 mA. Data were processed with BIOTEX (Molecular Structure Corporation, Inc.). The structure was solved by molecular replacement (MR) using data from 8.0- to 4.0-Å resolution and the 1 L chain from antibody HC19 23, 1GIG, as the search model. The MR was carried out using EPMR 24, which resulted in a solution with a correlation coefficient of 0.372. Search models using structures of several antibody H chains failed to produce a solution. Rigid body refinement of the L chain using TNT 25, using data from 10.0 to 3.0 Å, reduced the R factor to 44.6%. Phases from this partial structure were used to calculate an electron density map that revealed some interpretable density for the H chain. The D1.3 H chain 26, 1VFA, was then manually rotated into the density, and rigid body refinement was carried out. This reduced the R factor to 35.6%, at which point the correct side chains were substituted in the H chain. Positional (xyz) refinement, using data from 10.0 to 2.8 Å, was then initiated, and reduced the R factor to 22.9%. The resulting electron density map revealed very clear density for the NPPC hapten, which was then included, and the resulting model was refined via xyz, and subsequently via positional and thermal parameter (xyzb) refinement. xyzb refinement was carried out initially using data from 10.0- to 2.6-Å resolution. The final refinement included data extending from 10.0- to 2.35-Å resolution. The current structure includes residues 1–109 of the L chain, 1–112 of the H chain, the NPPC molecule, and 88 solvent molecules. PROCHECK analysis 27 revealed 97.9% of residues in allowed regions (78% in most favored), and 2.1% in generously allowed regions. See Table for selected crystallographic refinement statistics. Coordinates have been deposited in the Protein Data Bank with accession code 1DL7.
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2b, 1) is a member of a set of clonally related antibodies produced in a secondary response to PC–protein. High affinity for the hapten analogue NPPC is attributable to mutations at positions 52, 53, and 55 in CDR2 of the 1 L chain 16. Mutations at these key positions occur repeatedly in M3C65 clonal relatives, as well as in unrelated -bearing anti–PC protein hybridomas, indicating that LCDR2 replacements are vital to expansion and selection of these memory antibodies 1617. To assess whether the hapten–carrier linkage beyond the diazophenyl moiety forms part of the immunogenic structure of PC–KLH, we have analyzed binding to a number of structures representing distinct carrier epitopes. We also determined whether these structures are recognized differently as mutations accumulate. A comparison was made of the affinity of antibody constructs expressing zero (germline), one, or two mutations with the binding of M3C65, which contains all three pertinent mutations in LCDR2. The sequences of the H and L chain V regions and the positions of mutations are indicated in Fig. 1. The PC ligands used to measure affinity by fluorescence quenching are shown in Fig. 2.
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PC–tyrosine > NPPC, with the affinity for PC–histidine about ten times that of the affinity for NPPC. Although the affinity for NPPC increased as mutations accumulated in the L chain, all the mutant antibodies had higher affinity for PC–tyrosine and PC–histidine compared with antibody expressing germline L chain. It is noteworthy that ligand recognition shifts toward PC–tyrosine with the accumulation of mutations in the M3C65 antibody. Thus, binding to PC–tyrosine or PC–histidine allows for detection of changes in fine specificity, with the affinity of the mutated M3C65 antibody for PC–tyrosine being fivefold greater than for PC–histidine. These results demonstrate that carrier determinants, as represented by tyrosine and histidine, contribute to binding, and that somatic mutation can modulate carrier recognition.
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105 times more NPPC than PC–BSA was required to reduce binding by 50% 16. Is the superior inhibitory capacity of PC–protein due solely to the effects of avidity, or does effective binding correlate with an increase in size of the carrier? To answer these questions, we determined the intrinsic affinity of antibody for PC coupled to carriers of varying sizes. The tripeptide and the 15–amino acid peptide each have only one tyrosine (and no histidine) in the sequence, and are monosubstituted with PC (discussed in Materials and Methods), whereas the histones have multiple tyrosine and histidine residues 30. The results in Table show that affinity of antibody expressing germline 1 for the PC–GYA tripeptide is higher than for PC–tyrosine, PC–histidine, or PC–histone. These findings indicate that neither size nor avidity alone provides the advantage, but that qualitative characteristics of each individual carrier can be critical. After the introduction of the single mutation at position 53, binding to the monosubstituted 15–amino acid PC–peptide and to PC–histone is at levels near or equal to the fully mutated antibody. Thus, it appears that an increase in size of the carrier would beneficially affect binding to germline-encoded antibodies up to a certain point (PC–GYA > PC–histidine > NPPC), but a further increase in size represented by the protein conjugate, PC–histone, confers little affinity advantage until mutation occurs. The cumulative effects of somatic mutation on binding of small haptens in several systems indicate that high affinity can result from incremental improvements as mutations accumulate 3132. In M3C65, acquisition of high affinity for NPPC, PC–tyrosine, or PC–histidine by introduction of L chain mutations follows a similar pattern (Table ). In contrast, introduction of a single mutation at LCDR2 position 53 accounts for high-affinity binding of the monosubstituted 15–amino acid PC–peptide and results in a 200-fold increase in affinity for PC–histone. Thus, binding to the haptenic compounds mimics the stepwise acquisition of high affinity for small haptens seen with other antibodies, whereas high affinity to the larger PC–carrier structures may result from a single substitiution. Production of a high-affinity antibody by a single mutation emphasizes the adaptive potential of the group II combining site 33. The surprising difference in the effect of mutation on binding of the various PC-containing compounds raises a cautionary note concerning the extrapolation of antihapten binding results to the in vivo situation, where the complete hapten conjugate may be the structure relevant to shaping of the immune repertoire. In addition, as demonstrated here and by others, somatic mutation not only increases affinity for hapten, but may also shift antigenic specificity 343536.
Structure Determination and Overall Structure of the M3C65 sFv–NPPC Complex.
To determine the molecular basis of PC–carrier recognition and the contribution of somatic mutations, the crystal structure of an M3C65 sFv fragment complexed with NPPC was determined at a resolution of 2.35 Å (atom labeling of NPPC shown in Fig. 2 A). The sFv fragment of M3C65 was used for crystallographic analysis because the sFv binding data were in good agreement with that of intact antibody (Table ). Attempts to analyze the sFv fragment bound to the higher-affinity ligand, PC–tyrosine, were unsuccessful because the complex did not yield data-quality crystals. The crystallographic data collection and refinement statistics for the sFv–NPPC complex are shown in Table .
The combining site of M3C65 is a long narrow groove, 15 Å long by 5 Å wide by 7 Å deep, created by residues from LCDR1, LCDR2, LCDR3, HCDR2, and HCDR3 (Fig. 3). The binding pocket is unusual in that it is enclosed at one end by LCDR2 residue Thr55, which seals off the p-nitrophenyl group of the hapten from solvent. Contact with LCDR2 is rare in antibodies recognizing small haptens 18, but has been seen in the anti-DNP antibody ANO2, which has a similarly shaped combining site 37. In addition to the steric complementarity of the binding pocket to the hapten, electrostatic components appear to be crucial for selective binding and orientation. Specifically, at one end of the combining site, a negatively charged patch created by H chain CDR3 residue Asp95 attracts and orients the positively charged trimethylammonium portion of the choline moiety of NPPC (Fig. 3).
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interaction between the positively charged nitrogen of the choline moiety and the indole ring of Trp96. Cation- complexes have been shown to be important in other biological systems, including a group I PC-binding antibody M603, where these interactions with aromatic amino acid side chains enhance recognition of choline-containing ligands 39.
LCDR2 is the site of the three mutations at positions 52, 53, and 55 that confer increased binding affinity, and the crystal structure reveals the key roles of His53 and Thr55 in the formation of the combining site. His53 forms two hydrogen bonds, via the side chain N
1, to the carbonyls of L chain Gly49 (N1-O, 3.1 Å) and Gly50 (N1-O, 3.1 Å). These hydrogen bonds stabilize the combining site and anchor the side chain of His53 in a position to stack against and interact with NPPC. The side chain of His53 directly contacts the p-nitrophenyl group of NPPC (Table ), and also stacks against its aromatic group. The critical role played by His53 corroborates the finding that CDR2 position 53 is a specificity-determining residue in L chains 40.
Residue Thr55 plays two key roles in the architecture of the binding site near the p-nitrophenyl group. First, this β-branched residue closes off the end of the binding groove, allowing a tighter fit with the ligand by exclusion of water from the site, which is exposed in antibodies using the germline L chain. Second, Thr55 engages in a hydrogen bond from the O atom to the carbonyl oxygen of H chain Gly97. This interaction locks the L and H chains together in the specific conformation required for tight binding, and is therefore critical. Notably, this interaction is made possible by the presence of a proline at H chain position 98, which has adopted a cis conformation.
The role of mutated residue Lys52 is less clear, as the side chain is oriented away from the binding pocket. However, comparison of the M3C65 structure to the germline structure suggests that the importance of this mutation may lie in the replacement of the germline asparagine residue. In the germline 1 structure, Asn52 hydrogen bonds to Asn53. Such a hydrogen bond would also be possible between germline Asn52 and the mutated M3C65 His53 side chain, a contact that would disrupt the crucial contacts provided by His53. In contrast, a lysine at position 52 would be unlikely to hydrogen bond to His53, allowing His53 to make critical architectural contacts necessary for optimal binding of NPPC. Mutation away from asparagine may be aided by the high mutability index of the germline codon sequence 41.
The structural analyses of several other antibody–hapten interactions indicate that amino acid replacements resulting from somatic mutation may directly affect contact residues, but more frequently elicit their effects indirectly via conformational changes in the binding site 4243444546. However, in M3C65, both effects are seen. The His53 and Thr55 replacements, along with the indirect effects of the Lys52 replacement, optimize and fix the shape of the combining cavity, whereas His53 also contacts the hapten directly.
Comparison of M3C65 1 Structure with Germline 1.
To assess structural changes in combining site shape resulting from somatic mutation, we compared the M3C65 antibody with that of an unmutated 1 structure. Superpositioning of hybridoma HC19 germline 1 23 with the mutated M3C65 sFv shows that the C backbone tracings are quite similar, with a root mean square deviation of 0.50 for 108 corresponding residues (Fig. 5). Strikingly, the only notable differences between the two structures are observed in LCDR2 and involve residues 52–56, the region containing the mutated residues, with the major shifts occurring in residues Thr55 and Pro56. As noted, this significant shift results from contacts made by the mutated LCDR2 residues His53 and Thr55, thus providing the necessary conformation of the hapten binding pocket. The C backbone tracings of germline 1 and M3C65 1 are very similar. However, one notable shift in amino acid side chain placement occurs in the binding pocket, where L chain residue Tyr32 has rotated 90° from the germline, orienting the ring face so that it is more perpendicular to the ligand. This rotation of Tyr32 also places the C of Tyr32 closer to the O4 of the ligand phosphate group, a shift from 4.0 to 2.8 Å, such that its positive edge complements the negatively charged phosphate group. This close approach is indicative of an unusually strong C-H···O hydrogen bond 47.
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Repertoire Shift in the Immune Response to PC–KLH.
Immunization of BALB/c mice with PC–KLH leads to expression of two antibody populations differing in fine specificity that predominate in the immune response at different times 712131415. This repertoire shift occurs in the late primary and early secondary response, resulting in loss of initial dominance by antibodies bearing the T15 idiotype (group I) and their replacement by group II antibodies that employ different VH and VL combinations. We propose that the new gene combinations employed by group II antibodies allow recognition of complex PC–carrier epitope(s), and that the ability of these combining sites to be shaped by favorable somatic mutations allows emergence and dominance by group II–specific B cells.
T15 antibodies are restricted to use of the VH1/V
22 gene combination. These antibodies undergo little affinity maturation 50515253, and mutated T15 antibodies generally have reduced ability to bind antigen 5054. It has been suggested that deleterious mutation of primary antibodies contributes to loss of dominance in the memory response 55. However, group II V regions appear to be equally susceptible to harmful mutations 5657. Thus, T15 may be unique in the failure of mutations to improve antigen binding 33.
Group II antibodies use a variety of H and L combinations unrelated to T15 7. Although the L chain is expressed in many memory anti–PC–KLH antibodies, -bearing antibodies can dominate (>75%) some responses. To clarify factors conferring selective advantage to these antibodies, we focused on the antigen binding properties and combining site structure of a prototype group II memory antibody, M3C65. We demonstrate that (a) carrier determinants extending from PC contribute to the affinity of M3C65, (b) the shape of the M3C65 combining site is well suited for accommodation of PC in the context of carrier residues, (c) somatic mutation can alter the specificity for carrier determinants, and (d) somatic mutation can increase affinity directly by affecting residues in the combining site that contact ligand and indirectly by modifying the shape of the combining site.
More than 35 years ago, it was suggested that antibodies elicited to conjugated haptens might recognize carrier residues in addition to the haptenic determinant 58. Defining the complete antigenic determinant has been difficult, but in many systems it is thought that the protein carriers of haptens make a significant contribution to binding by antihapten antibodies 374459. However, T15 has approximately equal intrinsic affinities for PC (Ka = 2.9 x 105 M–1) and NPPC (Ka = 1.8 x 105 M–1), indicating the absence of a requirement for the diazophenyl linkage between PC and the protein carrier 6061. These results are in agreement with the earlier conclusion that T15 antibodies recognize only the PC portion of DPPC, another hapten that mimics the linkage between PC and protein 13. Thus, the carrier linkage does not appear to be an essential part of the epitope, in contrast to group II antibodies, where the carrier linkage is an essential feature.
Comparison of Group I and Group II Combining Sites.
Inspection of the binding pockets of two group I antibodies, T15 4362 and the related antibody M603 6364, reveals structural differences that would allow better accommodation of carrier residues by group II antibodies. Fig. 6 shows comparative models of T15 bound to PC and M3C65 bound to NPPC. In the T15 combining site, PC is bound in a deep pocket anchored by contacts between the antibody and the phosphate group. The cavity of T15 is deeper than M3C65 as a result of longer LCDR1, HCDR2, and HCDR3 loops that protrude farther from the binding pocket. PC occupies only a small portion of the combining site, leaving room for carrier residues; however, the deeper wedge-shaped cavity may hinder accommodation of larger carrier structures. Because of the orientation of PC toward the H chain, the HCDR2 region of T15 would likely interact with carrier epitopes, but the extended length and protrusion of CDR2 may also restrict the carrier interactions by steric hindrance. Although recognition of carrier epitopes can be modulated by mutation of the T15 HCDR2 loop, the effect on binding is subtle 65. The shape of the M3C65 binding site appears to be more suited for binding PC-associated carrier residues than that of T15, and selected mutations may favor such interactions. The elongated shallow groove of the M3C65 combining site provides a flatter surface more akin to antibodies recognizing proteins than the deep pockets of many antibodies recognizing small antigens 184666. Extension of carrier residues would continue over the surface of the antibody in such a way as to offer potential interaction with other residues of LCDR2. The more ideal shape of the M3C65 binding site for accommodating carrier compared with T15 is reflected in the 1,000-fold better binding of PC–protein 716. Thus, the basic geometry of the combining sites differs between these two primary and secondary anti-PC antibodies, unlike the response to phenyloxazolone, where it has been postulated that crucial contact residues would maintain the same mode of binding after recruitment of a new VH 67.
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It has been postulated that repertoire shift could be driven by kinetic parameters, such that the hydrophobic and charge characteristics imparted to antibodies by new VH/VL gene combinations provide high association rates leading to more efficient long-range attraction of antigen 376. In addition, a combining site that is more accessible to antigen can lead to faster binding kinetics 4877. It will be interesting to determine the kinetic rates of the M3C65 mutant antibodies, as well as those of other group II antibodies, for comparison with the T15 antibody. These comparisons will allow an evaluation of the effect of combining site shape and the consequences of somatic mutation on binding kinetics of antibodies participating in repertoire shift in the anti–PC–KLH response.
The crystal structure of the M3C65 sFv has allowed us to correlate binding with structural features of the antibody that confer advantage to mutated antibodies forming the group II memory pool. The role of somatic mutation in optimizing binding site geometry during affinity maturation has been studied in previous crystallographic analyses of antibody–hapten complexes 44677879. However, the structural basis of repertoire shift has remained more elusive because of the lack of crystal structures representing different stages of the maturing antibody response. Comparison of T15 and M3C65 binding sites illustrates how the accommodation of carrier residues provides a mechanism for the repertoire shift seen in the immune response to PC–KLH.
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This work was supported in part by National Institutes of Health grants AI-14985 and AI-26827 to M.B. Rittenberg and grant GM-49244 to R.G. Brennan. M.A. Schumacher is a 1999 recipient of a Burroughs Wellcome Career Development Award.
Submitted: 18 February 2000
Revised: 25 April 2000
Accepted: 3 May 2000
M. Brown and M.A. Schumacher contributed equally to this work.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eisen H.N. & Siskind G.W.. Variations in affinities of antibodies during the immune response, Biochemistry., 3, 1964, 996–1008.[Medline]
Cumano A. & Rajewsky K.. Clonal recruitment and somatic mutation in the generation of immunological memory to the hapten NP, EMBO (Eur. Mol. Biol. Organ.) J., 5, 1986, 2459–2468.[Medline]
Foote J. & Milstein C.. Kinetic maturation of an immune response, Nature., 352, 1991, 530–532.[Medline]
Wysocki L., Manser T. & Gefter M.L.. Somatic evolution of variable region structures during an immune response, Proc. Natl. Acad. Sci. USA., 83, 1986, 1847–1851.
Weigert M.G., Cesari I.M., Yonkovich S.J. & Cohn M.. Variability in the lambda light chain sequences of mouse antibody, Nature., 228, 1970, 1045–1047.[Medline]
Berek C. & Milstein C.. Mutation drift and repertoire shift in the maturation of the immune response, Immunol. Rev., 96, 1987, 23–41.[Medline]
Stenzel-Poore M.P., Bruderer U. & Rittenberg M.B.. The adaptive potential of the memory responseclonal recruitment and epitope recognition, Immunol. Rev., 105, 1988, 113–136.[Medline]
Manser T., Wysocki L.J., Gridley T., Near R.I. & Gefter M.L.. The molecular evolution of the immune response, Immunol. Today., 6, 1985, 94–100.
Bennett L. & Bishop C.T.. Structure of the type XXVII Streptococcus pneumoniae (pneumococcal) capsular polysaccharide, Can. J. Chem., 55, 1977, 8–16.
Lim P.L., Leung D.T.M., Chui Y.L. & Ma C.H.. Structural analysis of a phosphorylcholine-binding antibody which exhibits a unique carrier specificity for Trichinella spiralis, Mol. Immunol., 31, 1994, 1109–1116.[Medline]
McWilliam A.S., Stewart G.A. & Allen W.. Phosphorylcholine bearing components of some helminthic parasiteslocalization to parasite lipoproteins, Comp. Biochem. Physiol. B., 85, 1986, 627–633.[Medline]
Chang S.P., Brown M. & Rittenberg M.B.. Immunologic memory to phosphocholine. II. PC-KLH induces two antibody populations that dominate different isotypes, J. Immunol., 128, 1982, 702–706.[Abstract]
Rodwell J., Gearhart P.J. & Karush F.. Restriction in IgM expression. IV. Affinity analysis of monoclonal antiphosphorylcholine antibodies, J. Immunol., 130, 1983, 313–316.[Medline]
Wicker L.S., Guelde G., Scher I. & Kenny J.J.. Antibodies from the Lyb5-B cell subset predominate in the secondary IgG response to phosphocholine, J. Immunol., 129, 1982, 950–953.[Abstract]
Heusser C.H., Bews J.P.A., Abersold R. & Blaser K.. Anti-phosphocholine antibodies with a preferred reactivity for either PC or PC phenyl represent independent expressions, Ann. Immunol., 135C, 1984, 123–129.
Brown M., Stenzel-Poore M.P., Stevens S., Kondoleon S.K., Ng J., Bachinger H.P. & Rittenberg M.B.. Immunologic memory to phospocholine keyhole limpet hemocyaninrecurrent mutations in the 1 light chain increase affinity for antigen, J. Immunol., 148, 1992, 339–346.[Abstract]
Stenzel-Poore M.P. & Rittenberg M.B.. Unequal distribution of replacement mutations in and light chains and their associated H chainsthe group II antibody response to phosphocholine-KLH, Steele E.J.. Somatic Hypermutation, 1991, 95–104, CRC Press, Boca Raton, FL.
Wilson I.A. & Stanfield R.L.. Antibody-antigen interactions, Curr. Opin. Struct. Biol., 3, 1993, 113–118.
Boss M.A., Kenten J.H., Wood C.R. & Emtage J.S.. Assembly of functional antibodies from immunoglobulin heavy and light chains synthesized in E, Coli. Nucleic Acids Res., 12, 1984, 3791–3806.
Barbar E., Martin T.M., Brown M., Rittenberg M.B. & Peyton D.H.. Binding of phenylphosphocholine-carrier conjugates to the combining site of antibodies maintains a conformation of the hapten, Biochemistry., 35, 1996, 2958–2967.[Medline]
Moulton H.M., Structure and specificity of the binding site of the anti-phenylphosphocholine antibody M3C65interactions with haptens and hapten-carrier conjugates. Ph.D. thesis, 1996, Portland State University, , Portland, ORpp. 197.
Chang S.P. & Rittenberg M.B.. Immunologic memory to phosphorylcholine in vitro. I. Asymmetric expression of clonal dominance, J. Immunol., 126, 1981, 975–980.[Medline]
Bizebard T., Mauguen Y., Skehel J.J. & Knossow M.. Use of molecular replacement in the solution of an immunoglobulin Fab fragment structure, Acta Crystallogr., B47, 1991, 549–555.
Kissinger C.R., Gehlaer D.K. & Fogel D.B.. Rapid automated molecular replacement by evolutionary search, Acta. Crystallogr., D55, 1999, 484–491.
Tronrud D.E., TenEyck L.F. & Matthews B.W.. An efficient general purpose least-squares refinement program for macromolecular structures, Acta. Crystallogr., A43, 1985, 489–501.
Bhat T.N., Bently G.A., Boulot G., Greene M.I., Tello D., Dall'Acqua W., Souchon H., Schwarz F.P., Mariuzza R.A. & Poljak R.J.. Bound water molecules and conformational stabilization help mediate an antigen-antibody association, Proc. Natl. Acad. Aci. USA., 91, 1994, 1089–1093.
Laskowski R.A., MacArthur M.W. & Thornton J.M.. PROCHECKa program to check the sterochemical quality of protein structures, J. Appl. Crystallogr., 26, 1993, 283–291.
Gelewitz E.W., Riedeman W.L. & Klotz I.M.. Some quantitative aspects of the reaction of diazonium compounds with serum albumin, Arch. Biochem. Biophys., 53, 1954, 411–423.
Swerdlow R.D., Ebert R.F., Lee P., Bonaventura C. & Miller K.I.. Keyhole limpet hemocyaninstructural and functional characterization of two different subunits and multimers, Comp. Biochem. Physiol., 113B, 1996, 537–548.
von Holt C., Brandt W.F., Greyling H.J., Lindsey G.G., Retief J.D., Rodrigues J.A., Schwager S. & Sewell B.T.. Isolation and characterization of histones, Methods Enzymol., 170, 1989, 503–522.
Kocks C. & Rajewsky K.. Stepwise intraclonal maturation of antibody affinity through somatic hypermutation, Proc. Natl. Acad. Sci. USA., 85, 1988, 8206–8210.
Sharon J.. Structural correlates of high antibody affinityThree engineered amino acid substitutions can increase the affinity of an anti-p-azophenylarsonate antibody 200-fold, Proc. Natl. Acad Sci. USA., 87, 1990, 4814–4817.
Chen C., Roberts V.A., Stevens S., Brown M., Stenzel-Poore M.P. & Rittenberg M.B.. Enhancement and destruction of antibody function by somatic mutationunequal occurrence is controlled by V gene combinatorial associations, EMBO (Eur. Mol. Biol. Organ.) J., 14, 1995, 2784–2794.[Medline]
Giusti A.M., Chien N.C., Zack D.J., Shin S.-U. & Scharff M.D.. Somatic diversification of S107 from an antiphosphocholine to an anti-DNA autoantibody is due to a single base change in its heavy chain variable region, Proc. Natl. Acad. Sci. USA., 84, 1987, 2926–2930.
Hande S. & Manser T.. Single amino acid substitutions in V(H) CDR2 are sufficient to generate or enhance the specificity of two forms of an anti-arsonate antibody variable region for DNA, Mol. Immunol., 34, 1997, 1281–1290.[Medline]
Kussie P.H., Parhami-Seren B., Wysocki L.J. & Margolies M.N.. A single engineered amino acid substitution changes antibody fine specificity, J. Immunol., 152, 1994, 146–152.[Abstract]
Brünger A.T., Leahy D.J., Hynes T.R. & Fox R.O.. 2.9 Å resolution structure of an anti-dinitrophenyl-spin-label monoclonal antibody Fab fragment with bound hapten, J. Mol. Biol., 221, 1991, 239–256.[Medline]
Nicholls A., Sharp K. & Honig B.H.. Protein folding and associationinsights from the interfacial and thermodynamic properties of hydrocarbons, Proteins., 11, 1991, 281–296.[Medline]
Doughtery D.A.. Cation- interactions in chemistry and biologya new view of benzene, Phe, Tyr, and Trp, Science., 271, 1996, 163–168.[Abstract]
Padlan E.A., Abergel C. & Tipper J.P.. Identification of specificity-determining residues in antibodies, FASEB J., 9, 1995, 133–139.[Abstract]
Shapiro G.S., Aviszus K., Ikle D. & Wysocki L.J.. Predicting regional mutability in antibody V genes based solely on di- and trinucleotide sequence composition, J. Immunol., 163, 1999, 259–268.
Jeffrey P.D., Strong R.K., Sieker L.C., Chang C.Y., Campbell R.L., Petsko G.A., Haber E., Margolies M.N. & Sheriff S.. 26-10 Fab-digoxin complexaffinity and specificity due to surface complementarity, Proc. Natl. Acad. Sci. USA., 90, 1993, 10310–10314.
Chien N.C., Roberts V.A., Giusti A.M., Scharff M.D. & Getzoff E.D.. Significant structural and functional change of an antigen-binding site by a distant amino acid substitutionproposal of a structural mechanism, Proc. Natl. Acad. Sci. USA., 86, 1989, 5532–5536.
Strong R.K., Petsko G.A., Sharon J. & Margolies M.N.. Three-dimensional structure of murine anti-p-azophenylarsonate Fab 36-71. 2. Structural basis of hapten binding and idiotypy, Biochemistry., 30, 1991, 3749–3757.[Medline]
Padlan E.A.. Anatomy of the antibody molecule, Mol. Immunol., 31, 1994, 169–217.[Medline]
Patten P.A., Gray N.S., Yang P.L., Marks C.B., Wedemayer G.J., Boniface J.J., Stevens R.C. & Schultz P.G.. The immunological evolution of catalysis, Science., 271, 1996, 1086–1091.[Abstract]
Derewenda Z.S., Lee L. & Derewenda U.. The occurrence of C-H...O hydrogen bonds in proteins, J. Mol. Biol., 252, 1995, 248–262.[Medline]
Milstein C. & Neuberger M.S.. Maturation of the immune response, Adv. Protein Chem., 49, 1996, 451–485.[Medline]
Linton P.J., Lo D., Lai L., Thornbecke G.J. & Klinman N.R.. Among naive precursor cell subpopulations only progenitors of memory B cells originate germinal centers, Eur. J. Immunol., 22, 1992, 1293–1297.[Medline]
Gearhart P.J., Johnson N.D., Douglas R. & Hood L.. IgG antibodies to phosphorylcholine exhibit more diversity than their IgM counterparts, Nature., 291, 1981, 29–34.[Medline]
Andres C.M., Maddalena A., Hudak S., Young N.M. & Claflin J.L.. Anti-phosphocholine hybridoma antibodies II. Functional analysis of binding sites within three antibody families, J. Exp. Med., 154, 1981, 1584–1598.
Claflin J.L., George J., Dell C. & Berry J.. Patterns of mutations and selection in antibodies to the phosphocholine-specific determinant in Proteus morganii, J. Immunol., 143, 1989, 3054–3063.[Abstract]
Chen C., Roberts V.A. & Rittenberg M.B.. Generation and analysis of random point mutations in an antibody CDR2 sequencemany mutated antibodies lose their ability to bind antigen, J. Exp. Med., 176, 1992, 855–866.
Claflin J.L. & Berry J.. Genetics of the phosphocholine-specific antibody response to Streptococcus pneumoniae, J. Immunol., 141, 1988, 4012–4019.[Abstract]
Kalinke U., Bucher E.M., Ernst B., Oxenius A., Roost H.-P., Geley S., Kofler R., Zinkernagel R.M. & Hengartner H.. The role of somatic mutation in the generation of the protective humoral immune response against vesicular stomatitis virus, Immunity., 5, 1996, 639–652.[Medline]
Wiens G.D., Heldwein K.A., Stenzel-Poore M.P. & Rittenberg M.B.. Somatic mutation in VH complementarity-determining region 2 and framework region 2differential effects on antigen binding and immunoglobulin secretion, J. Immunol., 159, 1997, 1293–1302.[Abstract]
Wiens G.D., Roberts V.A., Whitcomb E.A., O'Hare T., Stenzel-Poore M.P. & Rittenberg M.B.. Harmful somatic mutationslessons from the dark side, Immunol. Rev., 162, 1998, 197–209.[Medline]
Karush F.. Immunologic specificity and molecular structure, Adv. Immunol., 2, 1962, 1–40.
Källberg E., Gray D. & Leanderson T.. The effect of carrier and carrier priming on the kinetics and pattern of somatic mutation in the VOx1 gene, Eur. J. Immunol., 25, 1995, 2349–2354.[Medline]
Bruderer U., Stenzel-Poore M.P., Bachinger H.P., Fellman J.H. & Rittenberg M.B.. Antibody combining site heterogeneity within the response to phosphocholine-keyhole limpet hemocyanin, Mol. Immunol., 26, 1989, 63–71.[Medline]
O'Hare T. & Rittenberg M.B.. A simple method for determining KAs of both low and high affinity IgG antibodies, J. Immunol. Methods., 218, 1998, 161–167.[Medline]
Brown M., Rittenberg M.B., Chen C. & Roberts V.A.. Tolerance to single, but not multiple, amino acid replacements in antibody VH CDR2a means of minimizing B cell wastage from somatic hypermutation?, J. Immunol., 156, 1996, 3285–3291.[Abstract]
Segal D.M., Padlan E.A., Cohen G.H., Rudikoff S., Potter M. & Davies D.R.. The three dimensional structure of a phosphorylcholine-binding mouse immunoglobulin Fab and the nature of the antigen binding site, Proc. Natl. Acad. Sci. USA., 71, 1974, 4298–4302.
Satow Y., Cohen G.H., Padlan E.A. & Davies D.R.. Phosphocholine binding immunoglobulin Fab McPC603an X-ray diffraction study at 2.7 Å, J. Mol. Biol., 190, 1986, 593–604.[Medline]
Brown M., Wiens G.D., O'Hare T., Stenzel-Poore M.P. & Rittenberg M.B.. Replacements in the exposed loop of the T15 antibody VH CDR2 affect carrier recognition of PC-containing pathogens, Mol. Immunol., 36, 1999, 205–211.[Medline]
Amit A.G., Mariuzza R.A., Phillips S.E.V. & Poljak R.J.. Three-dimensional structure of an antigen-antibody complex at 2.8 Å resolution, Science., 233, 1986, 747–753.
Alzari P.M., Spinelli S., Mariuzza R.A., Boulot G., Poljak R.J., Jarvis J.M. & Milstein C.. Three-dimensional structure determination of an anti-2-phenyloxazolone antibodythe role of somatic mutation and heavy/light chain pairing in the maturation of an immune response, EMBO (Eur. Mol. Biol. Organ.) J., 9, 1990, 3807–3814.[Medline]
Berek C., Griffiths G.M. & Milstein C.. Molecular events during maturation of the immune response to oxazolone, Nature., 316, 1985, 412–418.[Medline]
Manser T., Wysocki L.J., Margolies M.N. & Gefter M.L.. Evolution of antibody variable region structure during the immune response, Immunol. Rev., 96, 1987, 141–162.[Medline]
Newman M.A., Mainhart C.R., Mallett C.P., Lavoire T.B. & Smith-Gill S.J.. Patterns of antibody specificity during the BALB/c immune response to hen eggwhite lysozyme, J. Immunol., 149, 1992, 3260–3272.[Abstract]
Clarke S.H., Staudt L.M., Kavaler J., Schwartz D., Gerhard W.U. & Weigert M.G.. V region gene usage and somatic mutation in the primary and secondary responses to influenza virus hemagglutinin, J. Immunol., 144, 1990, 2795–2801.[Abstract]
Goldbaum F.A., Cauerhff A., Velikovsky A., Llera A., Riottot M.-M. & Poljak R.J.. Lack of significant differences in association rates and affinities of antibodies from short-term and long-term responses to hen egg lysozyme, J. Immunol., 162, 1999, 6040–6045.
Roost H.P., Bachmann M.F., Haag A., Kalinke U., Pliska V., Hengartner H. & Zinkernagel R.M.. Early high-affinity neutralizing anti-viral IgG responses without further overall improvements of affinity, Proc. Natl. Acad. Sci. USA, 92, 1995, 1257–1261.
Foote J. & Eisen H.N.. Kinetic and affinity limits on antibodies produced during immune responses, Proc. Natl. Acad. Sci. USA., 92, 1995, 1254–1256.
Batista F.D. & Neuberger M.S.. Affinity dependence of the B cell response to antigena threshold, a ceiling, and the importance of off-rate, Immunity., 8, 1998, 751–759.[Medline]
England P., Nageotte R., Renard M., Page A.-L. & Bedouelle H.. Functional characterization of the somatic hypermutation process leading to antibody D1.3, a high affinity antibody directed against lysozyme, J. Immunol., 162, 1999, 2129–2136.
McManus S. & Riechmann L.. Use of 2D NMR, protein engineering, and molecular modeling to study the hapten-binding site of an antibody Fv fragment against 2-phenyloxazolone, Biochemistry., 30, 1991, 5851–5857.[Medline]
Lascombe M.-B., Alzari P.M., Poljak R.J. & Nisonoff A.. Three-dimensional structure of two crystal forms of Fab R19.9 from a monoclonal anti-arsonate antibody, Proc. Natl. Acad. Sci. USA., 89, 1992, 9429–9433.
Wedemayer G.J., Patten P.A., Wang L.H., Schultz P.G. & Stevens R.C.. Structural insights into the evolution of an antibody combining site, Science., 276, 1997, 1665–1669.
Kabat E.A., Wu T.T., Perry H.M., Gottesman K.S. & Foeller C., Sequences of Proteins of Immunological Interest, 5th edition, 1991, U.S. Department of Health and Human Services, , Bethesda, MDpp. 2,597.
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