Conceived and designed the experiments: MKG MT SZ-P X-PK. Performed the experiments: JS HL XJ X-HW CW TO BV LL. Analyzed the data: MKG MT TC X-PK. Contributed reagents/materials/analysis tools: MKG MT X-HW CW TO PN SZ-P X-PK. Wrote the paper: MKG.
Dr. Max Totrov is affiliated with MOLSOFT LLC. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.
Preferential usage of immunoglobulin (Ig) genes that encode antibodies (Abs) against various pathogens is rarely observed and the nature of their dominance is unclear in the context of stochastic recombination of Ig genes. The hypothesis that restricted usage of Ig genes predetermines the antibody specificity was tested in this study of 18 human anti-V3 monoclonal Abs (mAbs) generated from unrelated individuals infected with various subtypes of HIV-1, all of which preferentially used pairing of the VH5-51 and VL lambda genes. Crystallographic analysis of five VH5-51/VL lambda-encoded Fabs complexed with various V3 peptides revealed a common three dimensional (3D) shape of the antigen-binding sites primarily determined by the four complementarity determining regions (CDR) for the heavy (H) and light (L) chains: specifically, the H1, H2, L1 and L2 domains. The CDR H3 domain did not contribute to the shape of the binding pocket, as it had different lengths, sequences and conformations for each mAb. The same shape of the binding site was further confirmed by the identical backbone conformation exhibited by V3 peptides in complex with Fabs which fully adapted to the binding pocket and the same key contact residues, mainly germline-encoded in the heavy and light chains of five Fabs. Finally, the VH5-51 anti-V3 mAbs recognized an epitope with an identical 3D structure which is mimicked by a single mimotope recognized by the majority of VH5-51-derived mAbs but not by other V3 mAbs. These data suggest that the identification of preferentially used Ig genes by neutralizing mAbs may define conserved epitopes in the diverse virus envelopes. This will be useful information for designing vaccine immunogen inducing cross-neutralizing Abs.
Human monoclonal antibodies (mAbs) against the third variable domain (V3) of the HIV-1 gp120 envelope protein derived from HIV-1 infected individuals display the ability to neutralize primary isolates representing different clades
Anti-V3 mAbs display a broad range of cross-neutralizing activities depending on conserved elements in the V3 loop and other factors, including immunoglobulin (Ig) gene usage. A study of Ig variable genes of heavy chains (VH) used by a panel of human anti-V3 mAbs revealed a significantly altered and restricted pattern of VH gene usage when compared to other anti-HIV-1 mAbs
In the context of stochastic recombination of Ig variable genes and different pairings of the heavy and light chain genes, the dominance of one particular VH gene paired in a restricted fashion with specific light chain variable genes (VL) suggests the existence of a predetermined structure of the antigen-binding site which fits to a particular epitope. To test this hypothesis, we analyzed the crystal structure of five Fabs of VH5-51/VL lambda genes encoded anti-V3 mAbs in complex with various V3 peptides.
The results confirmed our hypothesis and showed that (a) the shape of the antigen-binding site is similar in the five VH5-51/VL lambda encoded V3 mAbs and is primarily formed by the CDR H1, H2, L1 and L2 domains, (b) the majority of the key contact residues of the mAbs are the same and germline-encoded, and (c) the epitopes of these V3 mAbs have a very similar 3D structure. Furthermore, (d) a single mimotope peptide which mimics this epitope is recognized by a majority of VH5-51 anti-V3 mAbs, but not by other non-VH5-51 derived mAbs. These results suggest that identifying Ig genes preferentially used by neutralizing anti-HIV-1 mAbs has the potential to indicate the presence of conserved epitopes/antigens in diverse virus envelopes, which can then be used to design an immunogen based vaccine which induces cross-neutralizing Abs.
Recent analysis of the Ig variable genes coding for the heavy chains showed that the VH5-51 gene segment was preferentially used by 18 of 51 (35%) anti-V3 mAbs (
# | mAb |
Isotype | IGHV | IGLV lambda | Virus subtype |
Country of origin | Reference |
1 | 257 | IgG1 λ | VH5-51 | 1-47 | B | USA |
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2 | 782 | IgG1 λ | VH5-51 | 1-47 | B | USA |
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3 | 908 | IgG1 λ | VH5-51 | 1-47 | B | USA |
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4 | 1006-15 | IgG1 λ | VH5-51 | 1-47 | B | USA |
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5 | 2219 |
IgG1 λ | VH5-51 | 1-47 | B | USA |
|
6 | 2483 |
IgG1 λ | VH5-51 | 1.47 | B | USA |
|
7 | 2456 | IgG1 λ | VH5-51 | 3-25 | B | USA |
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8 | 419 | IgG1 λ | VH5-51 | 6-57 | B | USA |
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9 | 838 | IgG1 λ | VH5-51 | 3-1 | B | USA |
|
10 | 4085 | IgG1 λ | VH5-51 | 1-47 | CRF02_AG | Cameroon |
|
11 | 2557 | IgG1 λ | VH5-51 | 3-1 | CRF02_AG | Cameroon |
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12 | 2558 | IgG1 λ | VH5-51 | 3-1 | CRF02_AG | Cameroon |
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13 | 3019 | IgG1 λ | VH5-51 | 3-1 | CRF02_AG | Cameroon |
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14 | 3694 | IgG1 λ | VH5-51 | 3-1 | H | Cameroon |
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15 | 3792 | IgG1 λ | VH5-51 | 3-1 | C | India |
|
16 | 3906 | IgG1 λ | VH5-51 | 3-1 | C | India |
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17 | 4022 | IgG1 λ | VH5-51 | 3-10 | C | India |
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18 | 4025 | IgG1 λ | VH5-51 | 3-10 | non-B | India |
|
All mAbs were produced from HIV-1-infected individuals using cellular methods as described
Virus subtype which infected the blood donors;
These two mAbs, 2219 and 2483, which are derived from the same donor, are unique (see CDR H3 sequences in
Forty-eight light chains of human anti-V3 mAbs were sequenced to determine VL gene usage. All 18 VH5-51 genes paired only with lambda variable genes and 14 out of 18 used two particular lambda genes, L1-47 (n = 7) and L3-1 (n = 7), while the remaining four mAbs used L3-10 (n = 2), L3-25 (n = 1) and L6-57 (n = 1) genes (
VH5-51 paired exclusively with VL lambda genes, mainly 1-47 and 3-1, while non-VH5-51 paired with 18 various VL gene segments, both kappa and lambda, with preferential usage of VL lambda 3-25. The latter paired mainly with the VH4-59 gene segment (see
We also analyzed the pairing of VH5-51 and VL genes on 11 Abs derived from single B cells of healthy individuals
The specific epitope shapes within the V3 crown targeted by five VH5-51 derived anti-V3 mAbs were determined by crystal structures of their Fabs in complex with three V3 peptides representing the sequences of MN, NY5 (clade B viruses) and consensus clade A. The following structure complexes were determined: mAb 1006-15/V3MN (1006/MN), mAb 2219/V3MN (2219/MN), mAb 2557/V3NY5 (2557/NY5), mAb 2258/V3MN (2558/MN), and mAb 4025/V3ConA (4025/ConA). Structures of three complexes, 1006/MN, 2219/MN and 2557/NY5, were used previously to analyze the conserved elements of the V3 region
Parameter | Fab/Peptide Complex | |
2558/MN | 4025/ConA | |
Data Collection | ||
Beamline | X4C, NSLS | X4C, NSLS |
Space group | C2 | P3121 |
Unit cell dimensions: | ||
a, b, c (Å) | 102.4, 73.1, 88.7 | 136.1, 136.1, 73.4 |
α, β, γ (°) | 90.0, 122.0, 90.0 | 90.0, 90.0, 120.0 |
Wavelength (Å) | 0.97854 | 0.97883 |
Resolution (Å) | 50-1.60 (1.63–1.60) | 40.0-2.0 (2.07–2.00) |
Completeness (%) | 99.2 (98.4) | 98.5 (87.9) |
Redundancy | 6.1 (6.1) | 8.7 (5.0) |
I/σ | 29.1 (4.2) | 11.9 (1.2) |
Rsym (%) |
5.4 (43.1) | 10.6 (61.6) |
Refinement | ||
Resolution (Å) | 17.2–1.6 | 39.3–2.0 |
Number of reflections | 72601 | 52488 |
Rwork (%) | 17.42 | 17.66 |
Rfree (%) | 20.30 | 21.34 |
Rmsd bond length (Å) | 0.006 | 0.007 |
Rmsd bond angles (°) | 1.602 | 1.149 |
Ramachandran analysis | ||
Favored (%) |
98.07 | 96.98 |
Allowed (%) |
1.69 | 3.02 |
Outliers (%) |
0.24 | 0.00 |
PDB Accession Code | 3UJI | 3UJJ |
Values for the highest resolution shell are given in parentheses.
Actual number of residues is indicated in parentheses.
The epitope targeted by each mAb within the V3 loop crown was defined by calculating the atomic contacts of the residues of the V3 peptide in the crystallographic complex with that of the Fab using the ICM molecular modeling software package (Molsoft LLC, La Jolla, CA)
The epitopes were defined by atomic interactions of the corresponding Fab fragments with the three V3 peptides in complex. (A) Contact residues in the V3 peptides for each epitope-Fab complex of the anti-V3 mAbs. The height of each letter (abbreviations representing various amino acids) is proportional to the contact area of the V3 residue with the Fab fragment. (B) The contact area (Å2) for each residue interacting with Fab. The color for each complex is the same as the label in panel (A).
mAb/peptide | 302 | 303 | 304 | 305 | 306 | 307 | 308 | 309 | 312 | 313 | 314 | 315 | 316 | 317 | 318 | 319 | 320 |
1006/MN | K |
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R |
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G | P | R |
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2219/MN | K |
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R |
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G | P | R |
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2557/NY5 |
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G |
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G | P | R |
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2558/MN | N | K |
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R |
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G | P | F |
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T | T | |||
4025/ConA |
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G | P | Q |
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A | T | |||||
Mean area |
2 | 17 |
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18 |
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21 | 18 | 0 | 22 | 0 |
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6 | 5 |
The residues of the V3 peptides with 10 Å2 or more buried surface area in complex with Fabs were calculated using ICM-Pro program
Mean contact area (Å2) per residue.
The different Fabs were bound to recently defined elements of the V3 crown: the “arch” at the tip (β-turn) of the V3 loop (P312–P315), the “circlet” composed of regions in the N- and C-terminal β-strands of the crown (P306–P309 and P316–P317), and the “‘band” at the N- and C-terminal ends of the V3 crown (P304–P305 and P318)
Thus, the specific epitopes targeted by five different, cross-reactive VH5-51 V3 mAbs have a similar 3D structure, consisting of seven key residues present in the N- and C-terminal sides of the V3 loop (
We hypothesized that the remaining 13 VH5-51 anti-V3 mAbs, which were not crystallized in complex with the V3 peptides, may recognize the same or similar V3 epitope. To investigate a larger panel of anti-V3 mAbs for their recognition of this particular V3 conformation, we designed a constrained peptide mimotope which would preserve the 3D of the VH5-51 derived mAbs 2219, 2557 and 1006-15, as recently described, but would not react with other anti-V3 mAb that recognize different V3 epitopes
This cyclic mimotope was biotinylated, immobilized on streptavidin-coated ELISA plates, and tested for reactivity with 48 anti-V3 mAbs whose gene usage was known
(A) Binding of the VH5-51- and non-VH5-51-derived V3 mAbs to the peptide mimotope. Monoclonal Abs were tested at a concentration of 10.0 µg/ml in plates coated with biotinylated mimotope at a concentration of 1.0 µg/ml. (B) Relative affinities of mAbs binding to the mimotope as measured by 50% of maximal binding (“half-max”) to plates coated with the biotinylated mimotope at a concentration of 1.0 µg/ml. Lower half-max values reflect higher relative affinities.
We hypothesized that the VH5-51 V3 mAbs may have a structurally similar shape of the antigen-binding site (ACS), as they recognize uniform V3 epitope. This was confirmed by the crystallographic analysis of five Fabs (1006-15, 2219, 2557, 2558 and 4025) in complex with three different V3 peptides (MN, NY5 and consensus A).
Structural alignments of the variable fragment of the heavy and light chains of five VH5-51 derived Fabs in complex with V3 peptides revealed that the CDRs H1, H2, L1 and L2 loops superimpose well with a root mean square deviation (RMSD) of <1 Å (
(A) A structural superimposition of the Cα atoms of the Fab variable domains of complexes 1006/MN (green), 2219/MN (cyan), 2557/NY5 (magenta), 2558/MN (yellow), and 4025/ConA (salmon). Note the domains of CDR H1, H2, L1 and L2 superimposed well, while that of CDR L3 and H3 is more structurally divergent. (B) Same superimposition as that in panel (A) but with the key contact residues of the mAbs labeled (all contact residues are shown in
The binding site is shaped like a cradle in which the V3 peptide, in the form of a β-hairpin, is bound (
Thus, the strong structural commonality in the antigen-binding site of all five crystallized mAbs is determined primarily by the CDR H1, H2, L1 and L2 domains. Upon binding of the Fabs to V3 peptides, only the CDR H3 domain was seen to move towards the antigen, while the remaining CDR domains are stable and exhibit the same backbone conformation as in unbound Fab (X-P. Kong et al., unpublished data). The differences in binding affinity to a designed mimotope (
We speculated that the similar shape of the antigen-binding site of VH5-51 anti-V3 mAbs implied that the residues interacting with the V3 peptide are at the same position for each mAb. Crystallographic analysis confirmed our hypothesis, with the key contact residues in the CDR H1 and H2 regions in all five mAbs; they include WH33 in H1 (with some variations at positions H31 and H32) and YH52, DH54 and DH56 in H2 (
mAb/peptide | CDR H1 | CDR H2 | CDR H3 | Ref. |
1006/NY5 |
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2219/MN |
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2557/MN |
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2558/MN |
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This study |
4025/ConA |
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This study |
The contact residues (bold) of mAbs with more than 10 Å2 buried surface area in complex with V3 peptide were calculated using ICM program
Similarly, the contact residues in the CDR L1 of light chains are also germline-encoded, although they differ since three VL genes were used by five VH5-51 mAbs. The two residues in mAbs 1006-15 and 2219–NL31 and YL32, the three residues in mAbs 2557 –DL30, KL31 and YL32, and the two residues in mAbs 2558 and 4025–KL31 and YL32 which reacted with the V3 peptides, are present in the corresponding germline sequences of lambda light chain genes (
Germline | mAb/peptide | CDR L1 | CDR L2 | CDR L3 | Ref. |
VL1-47 | 1006/MN |
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VL1-47 | 2219/MN |
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VL3-1 | 2557/NY5 |
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VL3-1 | 2558/MN |
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This study |
VL3-10 | 4025/ConA |
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This study |
In a similar fashion as in
The contact residues in CDR H3 in five crystallized Fabs are all different, ranging from two to eight residues (
The presence of the key contact residues in the four CDR domains (H1, H2, L1 and L2) at the same position and which are germline-encoded, except of those located in the CDR H3, suggest that the antigen-binding site has conserved backbone conformation.
The V3 peptides with the sequence of MN, NY5 clade B viruses and consensus clade A and in complex with Fabs share an almost identical backbone structure shown by close superimposition of their Cα atoms (
(A) Structural superimposition of the Cα atoms of the V3 peptides in the complexes with five VH5-51 mAbs: 1006/MN (green), 2219/MN (cyan), 2557/NY5 (magenta), 2558/MN (yellow), and 4025/ConA (salmon). (B) For comparison, a structural superimposition of the V3 peptides complexed with non-VH5-51 mAbs: 537/MN (grey), 447/W2RW020 (violet), 3074/MN (orange), and 268/MN (teal); the complex of Fab with V3 peptide, 2557/NY5 (magenta), is retained for comparison. (C) V3 peptides in the various crystals noted in B after superimposing the Fab structures onto that of Fab 2557 (shown as a semi-transparent surface). Note the drastically different ways of binding V3 for the non-VH5-51 mAbs.
This is in contrast with the backbone conformations of the V3 peptides in complex with Fabs encoded by non-VH5-51 genes (
From this data, it is apparent that the backbone conformation of the V3 peptide in complex with the Fab is fully adapted to, and imprints, the shape of its antigen-binding site which is encoded by particular pair of Ig genes.
Restricted usage of Ig gene segments is rarely observed among Abs due to the exceptional variability resulting from Ig gene rearrangements and VH and VL gene pairing. While the mechanism of preferential Ig genes selection, when it occurs, is not clear, we have concluded on the basis of our data that the VH5-51/VL lambda genes preferentially used by anti-V3 mAbs are related to a particular conserved epitope in the V3 region of the HIV-1 envelope.
The crystal structures of five VH5-51/VL lambda derived V3 mAbs in complex with V3 peptides revealed that each Fab interacted with key residues at the same seven positions in the crown of the V3 loop, despite the fact that the amino acids at these positions could vary. The observation that mAbs derived from unrelated individuals and infected with disparate HIV-1 strains use the same human Ig variable segment, react with the same key residues, and recognize a nearly identical overall conformation in the V3 crown suggest that while V3 is variable in sequence and structurally flexible, a common structure(s) is retained across strains. Our results further indicate that the mAbs utilizing the VH5-51/VL lambda genes possess similarly shaped antigen-binding sites that are highly suitable for the recognition of this common and conserved V3 conformation. Thus, preferentially used Ig genes indicate that antibodies encoded by these genes recognize a conserved epitope which can be used for design of HIV vaccine immunogen.
The conserved shape of the binding sites is reflected in the identical lengths of three CDRs, H1, H2 and L2, and minimal variation in the CDR L1 (
The antigen-binding site of the VH5-51 derived V3 mAbs has a very stable backbone conformation which was not changed when Fabs were complexed with different V3 peptides. A similarly stable structure of the binding site was recently observed in a mAb against the hepatitis B virus
The CDR H3 domain has the special function of determining the fine specificity in each antibody
We hypothesize that the structure of the antigen-binding sites in the naïve B cell germline-encoded B cell receptor and in the affinity-matured mAbs are similar, based on the observation that the key contact residues in the heavy and light chains of crystallized Fabs are germline-encoded (
We have shown that the V3 sequence-fusion proteins used as boosting immunogens after gp120 DNA priming were immunogenic and induced neutralizing Abs in rabbits
Collectively, we have observed in this study that the preferential usage of the VH5-51 gene paired with VL lambda genes in a restricted manner forms the antigen-binding site which allows recognition of a conserved V3 epitope. This data suggest that identification of dominantly used Ig genes by antibodies, including neutralizing anti-HIV-1 mAbs, may allow for the discovery of novel conserved epitopes. It may have practical implications for the design of a vaccine immunogen targeting antigenically diverse pathogens such as HIV-1.
The study was approved by the New York University School of Medicine Institutional Review Board. All subjects signed written approved informed consent forms prior to participating in the study.
Forty-eight human anti-V3 HIV-1 mAbs were tested in this study. Eighteen mAbs are encoded by the VH5-51 gene segment (
Two 23-mer V3 peptides representing the sequences of clade B MN (301
The messenger RNA was extracted from hybridoma cell lines producing human anti-V3 mAbs and reverse transcribed into cDNA using oligo dT primer. A homopolymeric dCTP tail was added to the 3′ end of the total cDNA with terminal deoxynucleotidyl transferase. The variable domain of the lambda light chain genes (VL) was amplified from poly-C tailed cDNA by PCR using deoxyinosine-containing anchor primer as forwards primer (5′RACE Abridged Anchor primer,
PCR amplification was performed using a cycling program of 2 min at 94°C, 35 cycles of 60 s at 94°C, 60 s at 55°C, and 120 s at 72°C, followed by 7 min at 72°C. Ethidium bromide-stained 0.8% agarose gels were used to visualize the PCR products. The bands of the appropriate size were excised and cleaned with GeneElute Minus EtBr Spin Column (Sigma) then were cloned into 2.1-TOPO TA cloning (Invitrogen).
For each chain, 6 to 12 independent clones were screened. The plasmids with the appropriate inserts were sequenced in both directions using the M13 primers. All sequencing reactions were performed at the Macrogen, Rockville, MD. The sequence data were analyzed using Pregap4, BioEdit software and the International ImMunoGene Tics (IMGT) information system (
A standard ELISA was used to determine the binding of mAbs to the mimotope as previously described
Crystallization of Fabs in complex with V3 peptides was carried out to obtain the atomic structures of the antigen combining sites of the mAbs and their relationship to VH and VL gene usage. Briefly, Fab of each mAb was prepared by papain digestion followed by purification using Protein A affinity columns (GE Healthcare) and size exclusion chromatography
Amino acid sequences of the variable fragment of the heavy chains (A) and light chains (B) of 18 human anti-V3 mAbs encoded by the VH5-51 gene segment. The sequences are aligned with their corresponding germlines (the sequences of the heavy chains were aligned only to one germline sequence of IGHV5-51*01 according to IMGT system). Kabat numbering is used along with the IMGT CDR domain definitions. Insertion codes are shown as lowercase letters in the numbering line, following the “base” residue number for that insertion (e.g. the first “a” in the heavy chain figure, which follows residue 52, is for residue 52A). For CDRs H3 and L3, insertion codes are represented as “x” to preserve the alignment. Dots indicate identity, while letters indicate substitutions in the heavy and light chains. CDR H3 and Framework 4 are not aligned to germlines.
(TIF)
Structures of light chains, heavy chains, and the V3 peptides in the V3-Fab complexes. The key contact residues of the Fabs in the light and heavy chains are labeled and numbered according to the standard Kabat numbering scheme.
(TIF)
Immunoglobulin gene usage for variable light chain of non-VH5-51 anti-V3 mAbs.
(DOC)
ELISA reactivity of VH5-51 and non-VH5-51 encoded anti-V3 mAbs with cyclic biotinylated mimotopes1.
(DOC)
We would like to thank Dr. Susanne Tranguch for critically reading the manuscript.