Structural Elucidation of the Mechanistic Basis of Degeneracy in the Primary Humoral Response

This information is current as Tarique Khan and Dinakar M. Salunke of October 2, 2021. J Immunol 2012; 188:1819-1827; Prepublished online 20 January 2012; doi: 10.4049/jimmunol.1102701 http://www.jimmunol.org/content/188/4/1819 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2012/01/20/jimmunol.110270 Material 1.DC1

References This article cites 41 articles, 13 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/188/4/1819.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on October 2, 2021 • Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Structural Elucidation of the Mechanistic Basis of Degeneracy in the Primary Humoral Response

Tarique Khan* and Dinakar M. Salunke*,†

The mechanistic basis for efficient combating of the infinite range of foreign Ags by the limited repertoire of naive Abs expressed on primary B cell surfaces during their first encounter was addressed through elegantly designed crystallographic analyses. Resolution of the discrepancy arising from the limited number of possible germline Ab receptors on primary B cells for recognizing the un- limited pool of possible Ags has been attempted by invoking the degenerate recognition potential of the germline Abs. Structural analyses of germline mAb BBE6.12H3 in an Ag-free state, as well as bound to four different peptide Ags, established the correlation of its degenerate specificity with conformational versatility of the paratope. Six distinct paratope topologies observed for a single germline mAb provided a quantitative description of the primary Ag recognition repertoire at the tertiary structural level. Each of the four different peptide Ags was bound specifically to a distinct conformation of the paratope, which was also different from that of the Ag-free states of the same germline mAb. A minimal conserved motif in the pristine Ag-combining site essential for multispe- Downloaded from cificity and Ag binding-mediated change in the elbow angle of Fab was also discernible. It is proposed that the generation of a pri- mary Ab repertoire involves large, yet finite, germline Ab clones, each capable of adopting discrete conformations, which in turn exhibit diverse binding modes. The Journal of Immunology, 2012, 188: 1819–1827.

he antigenic repertoire is infinite. To generate an effective of such polyreactive Abs (10). Thereafter, during secondary im- immune response against each of the possible Ags from mune responses, mature Abs with higher affinity and specificity http://www.jimmunol.org/ T this infinite pool, every Ag has to be specifically recog- for a foreign molecule can be evolved when a rapid change in nized in the initial encounter. Physicochemical principles of Ag concentration or presentation of the foreign molecule triggers a recognition would suggest that Abs required to neutralize the mutagenic proliferation of the germline Ab in question (11). None- infinite population of Ags have to be unlimited. This conundrum theless, crystallographic analysis of anti-p24 (HIV) mAb toward was elegantly answered many years ago by an expansion of the different analogs of a peptide epitope has demonstrated the poly- primary Ab repertoire through genetic recombination of VDJ gene specific potential of even a mature Ab (12). segments and by resultant combinatorial diversity arising from Indeed, the structural basis of Ag recognition has been addressed rearranged heterodimers of light and heavy chains (1). Recent through a vast number of crystallographic data generated on mature by guest on October 2, 2021 high-throughput sequencing data in zebra fish and humans have Ab–Ag complexes during the past three decades. Together, these provided further insights into the extent of the expressed Ab rep- Ab–Ag complex structures have provided snapshots of the immune ertoire, as envisaged by Tonegawa et al. (2, 3) However, quanti- recognition of diverse epitopes and offer physiological as well as tative assessment, even after considering other possible mecha- biophysical perspective to our understanding of Ag recognition. nisms, proves this diversity to be inadequate for recognition of However, initial encounters of foreign Ags by naive primary Abs infinite Ags (4). have not been adequately explored. We have sought to address the It was suggested that a degree of polyreactivity within a germline strategies needed for efficient recognition of the infinite range of Ab would provide a way by which a single Ab would be able to foreign Ags by the limited repertoire of naive primary Abs during recognize a range of Ags and so obviate the requirement for a their first encounter. singular Ab for every potential non-self immunogen (5–9). Even To understand the mechanistic basis of this event, we have the -driven modulations of epitopes could be handled carried out structural investigations on a polyreactive germline very efficiently by primary humoral immune defense through use mAb, BBE6.12H3 Fab. It is an immunologically well-characterized germline Ab derived from the major idiotypic response to the hapten (4-hydroxy-3-nitrophenyl)-acetate (NP) in C57BL/6 mice *Structural Biology Unit, National Institute of Immunology, New Delhi 110 067, (13). The L and H chain V region of germline mAb BBE6.12H3 India; and †Regional Centre for Biotechnology, Gurgaon 122 016, India has been shown to be constructed from Vl1, and from VH 186.2, Received for publication September 19, 2011. Accepted for publication December 6, D Fl16.1, and J 2 germline gene segments, respectively (Sup- 2011. H H plemental Fig. 3). Its mature counterpart Bg 53-5 contains 11 point This work was supported by the Department of Biotechnology, Government of India. , 1 in the L chain and the remaining 10 in the H chain The coordinates and structure factors presented in this article have been submitted to the Research Collaboratory for Structural Bioinformatics Data Bank (http:// (13). Through extensive kinetic and thermodynamic analyses, www.pdb.org) under accession codes 4a6y, 2y07, 2y06, 2y36, and 2xzq. BBE6.12H3 was shown to bind to a series of independent Address correspondence and reprint requests to Dr. Dinakar M. Salunke, Regional dodecapeptide Ags (5, 6). In the current study, four among those Centre for Biotechnology, 180 Udyog Vihar, Phase-I, Gurgaon 122016, India. E-mail peptide Ags—namely, B6-13, 3, BA7-09, and B6-07, which bind address: [email protected] to the germline mAb BBE6.12H3 with Kd values of 0.38 mM, 0.80 The online version of this article contains supplemental material. mM, 0.60 mM, and 0.26 mM, respectively (6)—were subjected to Abbreviations used in this article: NP, (4-hydroxy-3-nitrophenyl)-acetate; PEG, poly- crystallographic analysis. The binding affinities of these peptides ethylene glycol. were 30- to 100-fold higher than that of the cognate Ag (Kd = 23.0 Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 mM). Our data illustrate the importance of paratope adaptability www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102701 1820 DEGENERACY IN THE PRIMARY HUMORAL RESPONSE for facilitating polyreactivity of germline mAb through system- Refinement and model building atically designed crystallographic experiments. The present anal- Refinement was conducted with CNS (24). Both conventional Rwork ysis, in light of earlier studies (1, 5, 8, 9, 14–19), suggests the (crystallographic R-factor) and Rfree (free R-factor) (24) values were possibility that generation of diversity in the primary Ab repertoire monitored during the refinement. In each case, 10% of the total reflections may involve multiple mechanisms in a hierarchical order. The were set aside for calculation of Rfree values. Initially, rigid body refinement proposed model may account for enhanced diversity of primary was carried out for the complete Fab molecule, and subsequently, VH,VL, CH, and CL domains were treated as discrete units. The models were further immune receptors without increasing the size of the germline gene refined using the positional refinement protocol of CNS. Electron density repertoire. maps were displayed with the help of the program COOT (25). As the refinement progressed, the peptide models could be built into electron Materials and Methods densities within the corresponding Ag-combining sites. Water molecules Peptides were added using the water pick program in CNS. The quality of the model was checked with MolProbity (26). Structural models have been generated Peptides were essentially derived using the Phage Display Peptide Library with PyMOL (http://www.pymol.org), and the structural superimpositions Kit (New England Biolabs, Cambridge, MA), as previously reported (6). were accomplished using the program SUPERPOSE from the CCP4 suite Interactions between Ab and peptide and their thermodynamic analysis by (27). The epitope–paratope interactions and buried surface areas were an- surface plasmon resonance have also been previously reported (6). Inde- alyzed using PISA (28). pendent peptides selected from a panel of a random phage library binding to germline mAb (6) were used for cocrystallization experiments. The 12- Accession codes mer peptides were synthesized by the solid-phase method on an automated The coordinates and structure factors have been deposited in the Research peptide synthesizer (431A; Applied Biosystems, Foster City, CA). Cleav- Collaboratory for Structural Bioinformatics Protein Data Bank (http://www. age was performed using trifluoroacetic acid (Sigma-Aldrich, St. Louis, pdb.org) under the accession codes 4a6y, 2y07, 2y06, 2y36, and 2xzq. MO). Crude peptides were purified on a Delta Pak C18 column (Waters, Downloaded from Milford, MA), using a linear gradient of acetonitrile containing 0.1% tri- fluoroacetic acid. The purity of peptides was characterized by mass spec- Results trometry. Crystal structures of germline mAb BBE6.12H3 Abs The diffraction-quality crystals of BBE6.12H3 Fab, as well as its complexes with the peptide Ags B6-13, 3, BA7-09, and B6-07, The hybridomas used in this study were previously generated by following were crystallized using 21% polyethylene glycol (PEG; 3.3 a standard method (13). Hybridoma cells secreting IgG BBE6.12H3 were http://www.jimmunol.org/ suspended in 500 ml Dulbecco’s PBS and were injected into the peritoneal kDa), 21% PEG (8.0 kDa), 19% PEG (6.0 kDa), 17% PEG (6.0 kDa), cavity of male BALB/c mice gamma irradiated (400 rads) and primed with and 23% PEG (3.3 kDa) in the presence of ZnCl2, respectively. In the Freund’s incomplete adjuvant 72 h prior to injecting the hybridoma cells. current study, these peptides are referred to as Dlw, Gdp, Ppy, Yql, 5 6 A total of 5 3 10 –5 3 10 hybridoma cells were injected into each and their complexes with Fab as BBE6.12H3-Dlw, BBE6.12H3- mouse. Ascitic fluid could be tapped from the peritoneal cavity of mice after ∼4–5 d. The animal experiments were approved by the Institutional Gdp, BBE6.12H3-Ppy, and BBE6.12H3-Yql, respectively. The crys- Animal Ethics Committee. tal structures were determined at the 2.4–2.9 A˚ resolution range. Crystal data and refinement statistics for all five structures are Fab preparation shown in Table I. The L and H chains of all BBE6.12H3 structures

The IgG was purified from mouse ascitic fluid by 40% ammonium sulfate contained 211 and 220 residues, respectively. Electron density for by guest on October 2, 2021 precipitation followed by resuspension of the precipitate in 10 mM Tris (pH Ser136H–Asn140H was ambiguous in all Fab–peptide complexes, as 8.5). Further purification of the IgG was carried out by ion-exchange in many other reported Fab structures (29). chromatography, using a DEAE-5PW anion-exchange column. Fab frag- ments of the Ab were prepared by papain digestion of the purified IgG at pH The asymmetric unit of the native Fab contained two molecules 7.1, and the Fab was purified from the digestion mixture, using DEAE (BBE6.12H3-Native-AB and BBE6.12H3-Native-LH). The four anion-exchange chromatography. The purity was checked by SDS-PAGE, complexes with the dodecapeptides Dlw, Gdp, Ppy, and Yql and the concentration of the Fab was estimated by protein assay (Bio-Rad, belonged to the same space group, and each contained one mol- Hercules, CA). ecule per asymmetric unit (Table I). Therefore, five crystals gen- Crystallization erated a database of six Fab molecules. Models were built for the The diffraction data were collected from crystals grown in polyethylene first 10, 6, and 9 residues of the peptide Ags Dlw, Ppy, and Yql, glycol and 50 mM Tris buffer (pH 7.1). The native as well as the Fab-peptide respectively. Nine residues of Gdp peptide, starting at the second cocrystals were obtained by hanging drop vapor diffusion at 28˚C, using residue from the N terminus, were evident in the electron density a starting Fab concentration of 10 mg/ml. A 40-fold molar excess of the (see Table II). Electron densities for the remaining amino acids each peptide was used for cocrystallization experiments. were not visible even when the corresponding electron density Data collection maps were rendered at low s values. The electron density maps of the peptides observed in the structures of the Fab–peptide com- Diffraction data for the native Fab and its complex with the peptide Ppy were collected on a home source, RU300 (Rigaku, Japan). Data for the Fab plexes are shown in Supplemental Fig. 1. complexes with the peptides Dlw, Gdp, and Yql were collected using Ag-free Fab structures synchrotron (BM14, European Synchrotron Radiation Facility, Grenoble, France) at wavelengths 0.953, 0.973, and 0.953, respectively. The crystals Evaluating the structural flexibility of the germline Ab paratope by were cryoprotected by being soaked in mother liquor containing 25% superimposition of the variable domains of Ag-free BBE6.12H3 glycerol and were flash frozen. Data were integrated with MOSFLM (20) Fab structures revealed two distinct CDRH3 loop conformations and scaled with SCALA (21). (Fig. 1A). The remaining CDR loops of both H and L chains were Structure determination identical. The pairwise RMSD values for backbone and all atoms The structure of native Fab was determined by molecular replacement using of CDRs, shown in Supplemental Table I, also indicated major N1G9 Fab (Protein Data Bank code: 1NGQ) as the model in AMoRe (22). backbone movement in CDRH3. The Ag-free BBE6.12H3 Fab intensity data gave a good correlation coef- The surface features of CDRs of the two Ag-free Fab molecules ficient (CF = 50.9%), and subsequent refinement was made using this model. enhanced with hydropathy features are shown in Fig. 1B and 1C. The structures of Fab–peptide complexes were determined by molecular replacement, using PHASER (23), and the variable and constant domains Paratopes of these molecules exhibit dissimilar topologies and of native germline Ab Fab were used as separate ensembles. The corre- charge distribution. Both backbone conformations and side chain sponding models were subsequently refined. orientations contribute to these differences. The movement of The Journal of Immunology 1821

Table I. Data collection and refinement statistics

BBE6.12H3-native BBE6.12H3-Dlw BBE6.12H3-Gdp BBE6.12H3-Ppy BBE6.12H3-Yql Data collection Space group C2 C2 C2 C2 C2 Cell dimensions a, b, c (A˚ ) 86.3, 81.4, 128.9 56.0, 63.6, 113.4 55.5, 61.9, 112.5 54.4, 60.8, 111.7 55.9, 65.2, 113.6 b (˚) 90.5 90.4 90.6 94.8 90.2 Resolution (A˚ ) 50–2.9 50–2.7 50–2.5 50–2.4 38–2.4 Rmerge (%) 12.0 (42.0) 7.0 (28.0) 6.0 (23.0) 10.0 (34.0) 6.0 (17.0) (I)/(s I) 4.7 (2.5) 11.3 (4.1) 10.0 (2.5) 5.0 (3.6) 18.5 (7.9) Completeness (%) 93.5 (87.3) 100 (99.8) 99.9 (99.0) 95.5 (93.6) 99.3 (98.1) Multiplicity 3.0 5.1 4.0 6.2 6.2 Refinement Resolution (A˚ ) 50–2.9 30–2.7 30–2.5 30–2.4 30–2.4 No. reflections 16,819 9,995 11,673 13,136 16,061 Rwork/Rfree (%) 24.7/26.7 23.0/24.7 23.3/26.6 23.4/25.7 24.0/25.0 No. atoms Water 131 99 46 149 126 Protein 6463 3217 3223 3204 3233 Peptide — 77 65 53 74 B-factors (A˚ 2) Water 21.0 36.9 41.9 37.5 28.8 Downloaded from Protein 31.2 41.6 48.4 36.5 26.7 Peptide — 67.9 64.0 41.8 66.8 RMSDs Bond lengths (A˚ ) 0.009 0.009 0.008 0.009 0.008 Bond angles (˚) 1.7 1.9 1.7 1.7 1.8 Parameters in parentheses represent last resolution shell statistics. The single-letter amino acid names of three initial residues at the N terminus of the peptide have been used for naming the peptide in each case. http://www.jimmunol.org/

CDRH3 is manifested by displacement of the backbone by #5.71 A˚ CDRH3 (Fig. 2A). The loop conformations start to diverge at and by the rotation of several side chains (Fig. 1A). The RMSD of Tyr95H and do not realign until Phe100cH. This movement is ac- the superimposed native structures showed that Tyr95B and Tyr95H companied by displacement of the CDRH3 main chain by #7.68 differ in position by .6A˚ (measured between the side chain atoms). A˚ and by the rotation of several side chains (Fig. 2A). The CDRH2 also displayed variation in conformation, although to a lesser Comparative analysis of peptide Ag-bound Fab structures extent (Fig. 2A). The pairwise superimposition of the CDRs

Crystal structures of the BBE6.12H3 in complex with dodecapep- showed that the backbone of the CDRH3 in complexed isomers by guest on October 2, 2021 tide Ags provided insights into the mechanism by which germline deviates significantly (Supplemental Table I). mAbs can recognize and bind independent ligands. The CDRH3 The variable and constant domains of two native BBE6.12H3 Ab loop of mAb exists in different conformations in the absence of the structures are not in line with each other, as their elbow angles are Ag. We sought to investigate whether this conformational diversity 201.2˚ and 200.1˚, respectively. Elbow angles .180˚ have been was indeed exploited while binding to different Ags. Indeed, the Ca reported for the murine l-chain–bearing Fabs, even though elbow superimposition illustrated distinct conformations of CDRH3 in the angles range mostly from 130˚ to 180˚ (30). In peptide Ag-bound four Ag-bound structures (Fig. 2A). This dissimilarity was also per- structures, the two domains are in line with each other, and the ceptible from the diverse surface topologies of the Fab molecules elbow angles of Dlw-, Gdp-, Ppy-, and Yql-bound Fab structures in the Ag-bound form, as shown in Fig. 3. are 175.5˚, 175.9˚, 176.6˚, and 174.5˚, respectively (see Table II) Comparison of the CDRH3 loops in four complexes and in the (31). Thus, native Fab molecules, compared with Ag-bound Fab native state (Figs. 1A,2A) indeed shows that CDRH3 backbone structures, show differences in the relative orientations of the and side chain orientations construct a lid-like structure that variable and constant domains. The variable domain reorientations covers the Ag-binding pocket in native Fab molecules. In contrast, during complex formation with the peptides Dlw, Gdp, Ppy, and CDRH3 bends away from the hapten-binding cavity in all Ag- Yql induce movement of the VL and VH against each other, in bound structures. In all the peptide-bound Fab molecules, the comparison with native Fab molecules. Consequently, major binding site is exposed owing to this outward movement of changes occur in the elbow angles of complexed Fab fragments

Table II. Buried surface area and elbow angle analysis

˚ 2 ˚ 2 ˚ 2 ˚ 2 Complexed Peptides Peptide (A )VL (A )VH (A )VL–VH Interface (A ) Elbow Angle (˚) DLWTTAIPTIPS (BBE6.12H3-Dlw) 505 992 1147 2650 175.5 GDPRPSYISHLL (BBE6.12H3-Gdp) 492 1141 1213 2850 175.9 PPYPAWHAPGNI (BBE6.12H3-Ppy) 431 1049 1030 2509 176.6 YQLRPNAETLRF (BBE6.12H3-Yql) 536 1286 1043 3049 174.5 BBE6.12H3-Native-AB 1770.0 201.2 BBE6.12H3-Native-LH 1750.0 200.1 Buried surface areas were calculated with the program PISA (22), using a probe radius of 1.4 A˚ and van der Waals radii. Elbow angles were calculated using a Web server (26). All values for buried surface area were calculated for the modeled peptide structures (shown in bold letters). 1822 DEGENERACY IN THE PRIMARY HUMORAL RESPONSE

FIGURE 1. Comparative analysis of conformations of the CDR loop of Ag-free Fab molecules. A, Stereo view of superimposed CDRs of Ag-free molecules highlighting divergence in the CDRH3 conformation are shown as thin ribbons, and side chains of only CDRH3 loops are shown as sticks; BBE6.12H3-Native- AB, magenta; BBE6.12H3-Native-LH, cyan. Connolly surfaces of the Ag-free mAb BBE6.12H3 Fab molecules enhanced with the hydropathy feature are shown, in which the red to blue color spectrum represents hydro- phobicity to hydrophilicity, respectively. B,BBE6.12H3- Native-AB. C, BBE6.12H3-Native-LH. A box in B and C highlights the inward orientation of CDRH3 residues compared with outward movement of the same residues in Ag-bound structures, as shown in Fig. 3. Downloaded from http://www.jimmunol.org/ compared with Ag-free Fab. The largest change was found in the 2B). Their binding sites overlap. Three common residues—Trp33 case of BBE6.12H3-Yql. Comparing the different BBE6.12H3/ (CDRH1), Arg50 (CDRH2), and Trp91 (CDRL3)—form the shared peptide complexes with an Ag-free Fab fragment, a clear-cut cor- paratope (Fig 2A). For the conformation and relative orientations relation between the buried surface area at the VL–VH interface and of the different peptides, see Fig. 2B. These diverse conformations the extent of domain disposition was observed (Table II). of the peptides suggest topological nonequivalence between the bound epitopes and further confirm degeneracy of the germline Ab Interactions and conformations of the peptide Ags bound to toward the Ags that were remarkably different (Fig. 2B). A BBE6.12H3 comparison of the mode of binding of the four peptide Ags to All the peptides are located in and around a deep, hydrophobic BBE6.12H3 is shown in Fig. 3. All the four Ags were seen inter- by guest on October 2, 2021 pocket in different conformations, but in the same orientation (Fig. acting with an independent set of CDR residues (Fig. 3, Table IV).

FIGURE 2. Stereoscopic representation of superimposition of Ag-bound Fab molecules. A, CDRs of Ag-bound Fab molecules and side chains of all interacting residues are shown as thin sticks. Three common interacting residues— W33H (CDRH1), R50H (CDRH2), and W91L (CDRL3)—are shown as thick sticks. BBE6.12H3- Dlw, red; BBE6.12H3-Gdp, green; BBE6.12H3- Ppy, blue; BBE6.12H3-Yql, yellow. B,Onlypep- tide epitopes from the superimposed complexes are displayed as sticks for clear depiction of confor- mational distinctions between them. Dlw, red; Gdp, green; Ppy, blue; and Yql, yellow. The Journal of Immunology 1823 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 3. Comparison of paratope surfaces involved in Ag binding. Close-up view of Connolly surfaces of BBE6.12H3 Fab complexes with peptide Ags is shown (red to blue color spectrum represents hydrophobicity to polarity). The stereo view of the peptide Ag-bound states are shown, along with the critical interactions in each Fab/peptide complex; the complexed peptides (red sticks, N terminus at the right side) are orientated in the binding groove, with

VL at the left. The interacting residues and CDRs in each case are represented as green sticks and loops, respectively. Dotted lines indicate hydrogen bonds. A, Dlw, DLWTTAIPTIPS. B, Gdp, GDPRPSYISHLL. C, Ppy, PPYPAWHAPGNI. D, Yql, YQLRPNAETLRF. Bold letters indicate peptide residues, which were evident in electron density.

The buried surface areas for the different peptides, as well as for idues in the different structures were identical, except those of the Fab, VL,VH, and the VL–VH interface, given in Table II depicted residues from CDRH3 and CDRH2 (Fig. 2A). variations in the paratope topologies of BBE6.12H3 while binding The peptide Dlw lies in a twisted conformation at the interface of to independent peptides. In all complexes, the interactions were the variable domains of the H and L chains, and it predominantly specific, as many hydrogen bonds and salt bridge contacts were interacts with CDRL3 and CDRH2 residues (Fig. 3A, Tables III observed (Table III). In all, 22 residues of the Fab CDRs were and IV). Trp3 makes the most of the hydrophobic interactions by involved in interactions with the peptides in one case or another snugly fitting into the hydrophobic pocket (for residue-wise (Table IV). The side chain orientations of all the interacting res- interactions, see Table III and Fig. 3A). Details of the atoms in- 1824 DEGENERACY IN THE PRIMARY HUMORAL RESPONSE

Table III. Polar and side chain hydrophobic interactions between BBE6.12H3 and different peptides

BBE6.12H3 DLWTTAIPTIPS (Dlw) GDPRPSYISHLL (Gdp) PPYPAWHAPGNI (Ppy) YQLRPNAETLRF (Yql) CDR L1 Ser30b O Pro1 N Tyr32 Pro3, Pro5 Pro1, Pro2 Pro5 Tyr32 Oh Arg4 N CDRL3 Trp91 Leu2,Trp3 Pro5 Pro1, Pro2,Tyr3 Leu3, Pro5, Leu10 Trp91 N«1 Ser6 O Pro1 O Arg4 O, Pro5 O Tyr92 Leu3 Tyr92 N Leu3 O Ser93 N Leu3 O Ser93 Og Asp1 Od2 Gln2 O, Leu3 N, Arg4 N« CDRH1 Trp33 Trp3 Tyr7 Tyr3, Pro4,Trp6 His35 Trp3 CDRH2 Arg50 Nh1Tyr3 Oh Glu8 O Arg50 Nh2 Ser9 O His10 O Arg50 N« His10 O Thr57 N Ile7 O Thr57 O Pro8 N, Pro11 N Tyr59 Ile10

Tyr59 N Ile10 O Downloaded from Lys64 Nz Ile10 O Tyr95 Trp3 Tyr7 Trp6 CDRH3 Tyr95 Oh Ser6 Og Thr9 O Tyr97 Trp6 Side chain hydrophobic interactions were calculated using van der Waals radii in the contact program of CCP4 (15). Polar contacts were calculated using PISA (22). Salt bridges are indicated by bold text. http://www.jimmunol.org/ volved in polar interactions are given in Table III. The CDRH2 contacts between peptide and the Ab residues have been outlined. residues form most of the polar interactions; Thr57H makes three Dlw interactions include 135 van der Waals contacts with the Ab hydrogen bonds with three different peptide residues, namely, Ile7, residues. The common interacting residue Trp91L makes strong Pro8, and Pro11;Tyr59H makes a polar contact with Ile10. Another hydrophobic interactions with Leu2 and Trp3, whereas Trp33H CDRH2 residue, Lys64H, makes a salt bridge with Ile10 (Table III). contacts only Trp3. In addition to this, all three common interac- Only one residue of the L chain, Ser93L (CDRL3), interacts with ting residues make several van der Waals contacts with different Asp1 through a hydrogen bond. In Table IV, all the van der Waals peptide residues, as shown in Table IV. by guest on October 2, 2021

Table IV. Interactions of peptides with BBE6.12H3 Fab

Peptide Residues Ab Residues Dlw Gdp Ppy Yql Thr26L Tyr1 (5) Thr30aL Pro3 (5) Ser30bL Pro3 (5) Pro1 (11) Arg4 (10), Leu3 (1) Tyr32L Asp2 (1), Pro3 (8), Arg4 (4), Pro1 (2) Arg4 (3), Pro5 (4) Pro5 (6), Ser6 (13) Trp91L Leu2 (2), Trp3 (20) Pro5 (2), Ser6 (2) Pro1 (13), Pro2 (4) Arg4 (5), Leu3 (9), Pro5 (2), Tyr3 (6) Glu8 (2), Thr9 (15) Tyr92L Pro1 (4) Leu3 (13), Gln2 (12) Ser93L Asp1 (1) Pro1 (7), Pro2 (2) Leu3 (13), Arg4 (9), Pro5 (1) Asn94L Asp1 (2), Leu2 (9) Pro2 (2) Pro5 (3), Glu8 (3) His95L Gln2 (1) Trp96L Trp3 (2) Trp33H Trp3 (12), Thr4 (2) Tyr7 (8), Ser8 (4) Tyr3 (7), Pro4 (2), Trp6 (8) Glu8 (2), Thr9 (2) His35H Trp3 (3) Arg50H Leu2 (2), Trp3 (5) Thr4 (1), Ser9 (1), His10 (3) Tyr3 (8), Pro4 (1) Glu8 (3) Ile7 (1) Asp52H His10 (5) Ser54H His10 (1) Gly56H Ile7 (1), Pro8 (4) His10 (10) Thr57H Ala6 (1), Ile7 (6), Pro8 (8), Gly9 (3), Ile10 (4) Ala58H Leu2 (3), Pro8 (6), Ile10 (6) Tyr3 (2) Glu8 (5) Tyr59H Gly9 (2), Ile10 (6) Lys64H Ile10 (4) Tyr95H Trp3 (4) Trp6 (3) Thr9 (1) Tyr97H Tyr7 (1) Trp6 (2) Residues corresponding to those involved in NP binding in N1G9 Fab are italicized. The interacting paratope residues common in four peptide complexes are indicated by underlined text. The number of interactions of each Ab residue is shown in parentheses. The Journal of Immunology 1825

The peptide Gdp binds in extended conformation comprising binding site containing both conserved and nonconserved inter- three bends that form a wave-like stretch. The residues are pre- acting amino acids. Despite the extraordinary differences between dominantly in b conformation and show interactions with both cognate hapten Ag and the selected peptides, the Ag-binding H and L chain CDRs (Fig. 3B, Table IV). Pro3, Pro5, and Tyr7 con- pocket of the germline Ab could accommodate diverse peptide tribute to hydrophobic interactions (Table III). Atomic details of residues and also formed new hydrogen bonds (Table III). A the polar contacts are presented in Table III. Whereas Ser6, Ser9, schematic of the proposed model for the generation of primary Ab and His10 of Gdp interact with H chain residues, Tyr95H and diversity based on the present data and previous observations Arg50H via four hydrogen bonds, the side chains of residues Tyr32L (1, 15) is shown in Fig. 4. and Trp91L make polar contacts with Arg4 and Ser6, respectively (Fig. 3B, Table III). As illustrated in Table IV, 81 van der Waals Discussion contacts are formed with CDR residues. From the common in- Structural analyses involving a germline Ab that has not seen an Ag teracting residues, side chain atoms of only Arg50H make polar con- a priori demonstrated distinct conformational rearrangements to tacts with Ser9 and His10.Trp33H and Trp91L are involved in hy- recognize several structurally independent Ags. Six structures of drophobic contacts with Tyr7 and Pro5, respectively. As described the germline mAb BBE6.12H3, in different chemical environ- in Table IV, the common residues play a dominant role by con- ments, quantitatively define the conformational repertoire of the tacting a variety of peptide residues. CDRs, providing a rich data set for examining paratope topologies, Only six residues starting from the N terminus of the Ppy peptide although thermodynamic and computational studies have provided could be traced, as half of the peptide moved into the solvent and interesting clues regarding the generation of diversity in primary could not be mapped and the residues were presumably disordered. Ab response (5, 19, 33, 34). Similarly, crystallographic and kinetic Downloaded from The entire peptide is seen bound around the periphery of the Ag- analyses of mature Abs SPE7 and NQ22/61.1, respectively, have binding cavity by inserting a hydrophobic side chain into it. All the also suggested conformational isomerism (7, 19). Our data con- peptide residues, which were seen in electron density, are hydro- sisting of six BBE6.12H3 Fab structures—two Ag free and four phobic in nature; therefore, most of the binding energy comes from Ag bound—provided a rich source of information reflecting cor- hydrophobic interactions. These hydrophobic interactions were relation of paratope conformational flexibility and polyreactivity. observed mainly with the residues of CDRL1, CDRL3, CDRH1, In the case of germline mAbs 28B4, 48G7, and AZ28, confor- and CDRH3 (Fig. 3C, Table III). Nonetheless, two peptide resi- mational rearrangement of CDRH3 has been shown to facilitate http://www.jimmunol.org/ dues, Pro1 and Tyr3, make polar contacts with the side atoms of hapten binding. However, none of these studies have associated Ser30bL,Trp91L, and Arg50H, respectively. The details of the atoms conformational flexibility of CDRs with degenerate specificity involved in the polar contacts are illustrated in Table III. Trp91L (14, 35–37). and Trp33H have hydrophobic interactions with the three initial The observed conformational variations in germline Ab-com- residues and the last three residues of the Ppy peptide, respec- bining sites are consistent with the predictions based on thermo- tively. Trp91L,Trp33H, and Arg50H also contribute most of the van dynamic studies and molecular dynamics simulations (5, 33). On der Waals contacts. Details of these interactions are presented in the basis of energy considerations, the conformations adopted by Table IV. The peptide Ppy makes 78 van der Waals contacts.

CDRH3 cannot represent a continuum of conformational space by guest on October 2, 2021 The Ag Yql lies in the gully at the interface of CDRL1 and but, rather, correspond to a finite number of discrete states. The six CDRL3. It forms a hook-like structure by turning at residue Ala7, different observed conformations of CDRH3 and four slightly which anchors it into the Ag-binding cavity. Residue-wise inter- different conformations of CDRH2 suggest the possibility of cor- actions of Yql are depicted in Fig. 3D and Table IV. As shown in relation between the flexible nature of these loops and the para- Table III, the main chain oxygen atoms of peptide residues Gln2, tope recognition potential. Arg4, Pro5, Glu8, and Thr9 are hydrogen bonded to the side chains A correlation of the observed variability in CDR conformations of Ser93L,Trp91L, Arg50H, and Tyr95H, respectively. The oxygen with the polyspecificity of germline Ab has attractive physiological atoms of the peptide residue Leu3 make two polar contacts with implications. The pluripotency at a mature level can lead to serious the amide nitrogen of Tyr92L and Ser93L. Besides this, Leu3 and Arg4 form two more polar contacts with the hydroxyl of Ser93L.In this complex, unlike other three peptides, the majority of inter- actions are with the L chain and are electrostatic in nature. From the common interacting residues, Trp91L makes hydrophobic in- teractions with peptide residues. Trp33H is involved only in van der Waals interactions. Besides making van der Waals contacts, Arg50H also contributes to binding energy by making a hydrogen bond with the carboxyl oxygen atom of Glu8 (Table III). Residue- wise van der Waals interactions are shown in Table IV. Analysis of the structures of BBE6.12H3 in complex with the four synthetic peptide Ags revealed that the naive Ag-combining site could tolerate significant variability in the epitope (Fig. 2B). The residues of peptides, which bind at common paratope sites, FIGURE 4. Multiple mechanisms for enhancing primary Ab repertoire. were LWT (BBE6.12H3-Dlw), PSYIS (BBE6.12H3-Gdp), YPAW Germline Ab repertoire is generated at three levels in an amplified manner. (BBE6.12H3-Ppy), and PNAET (BBE6.12H3-Yql), and these In this schematic, the inverted pyramid represents the hierarchical contri- bution to the diversity of these stages. At the first level, the BCR repertoire residues have been depicted as dots in Supplemental Fig. 2. Three is generated during the development of B cells by somatic recombination common interacting residues, Trp33 (CDRH1), Arg50 (CDRH2), 91 of V, D, and J gene segments. The second level operates at the tertiary and Trp (CDRL3), form a minimal conserved motif essential for structure wherein each rearranged receptor exhibits diverse conformational recognition of multiple Ags (Fig. 2A). Interestingly, the same isomers. At the final level, each conformer can recognize independent residues were also shown to interact with the hapten (NP) (32). epitopes through alternative juxtapositions. The different colors represent Therefore, it can be said that germline Abs have a predesigned distinct Ab sequences. 1826 DEGENERACY IN THE PRIMARY HUMORAL RESPONSE immunological consequences. However, the pluripotency of ger- versity of a combinatorial antibody library gives insight into the human immu- noglobulin repertoire. Proc. Natl. Acad. Sci. USA 106: 20216–20221. mline Ab can contribute substantially to the enhancement of the pri- 4. Desiderio, S. V., G. D. Yancopoulos, M. Paskind, E. Thomas, M. A. Boss, mary immune receptor repertoire. All six conformational states of N. Landau, F. W. Alt, and D. Baltimore. 1984. Insertion of N regions into heavy- BBE6.12H3 represent an ensemble reflecting the conformational di- chain genes is correlated with expression of terminal deoxytransferase in B cells. Nature 311: 752–755. versity extant in the primary humoral response. Thus, the Ab–Ag 5. Manivel, V., N. C. Sahoo, D. M. Salunke, and K. V. Rao. 2000. Maturation of an complex structures provided snapshots of how the immune system antibody response is governed by modulations in flexibility of the antigen- could generate multiple specificities that allow for recognition of combining site. Immunity 13: 611–620. 6. Manivel, V., F. Bayiroglu, Z. Siddiqui, D. M. Salunke, and K. V. Rao. 2002. The diverse epitopes. Although these insights have arisen from studies primary antibody repertoire represents a linked network of degenerate antigen involving a germline Ab that binds to diverse 12-mer peptides, it specificities. J. Immunol. 169: 888–897. would be interesting to explore protein Ags as well. 7. James, L. C., P. Roversi, and D. S. Tawfik. 2003. Antibody multispecificity mediated by conformational diversity. Science 299: 1362–1367. The germline mAb BBE6.12H3 undergoes conformational 8. Nguyen, H. P., N. O. Seto, C. R. MacKenzie, L. Brade, P. Kosma, H. Brade, and changes in the CDRs while binding to independent Ags. In contrast, S. V. Evans. 2003. Germline antibody recognition of distinct carbohydrate the germline mAb 36–65 was seen to bind different Ags in a epitopes. Nat. Struct. Biol. 10: 1019–1025. 9. Yin, J., A. E. Beuscher, IV, S. E. Andryski, R. C. Stevens, and P. G. Schultz. similar conformation, essentially indicating that a rigid receptor 2003. Structural plasticity and the of antibody affinity and specificity. binding site can have different interactions with structurally dis- J. Mol. Biol. 330: 651–656. 10. Berek, C., and C. Milstein. 1987. Mutation drift and repertoire shift in the tinct Ag surfaces, without any conformational changes in the re- maturation of the immune response. Immunol. Rev. 96: 23–41. ceptor (15). In the unbound state, however, both the Abs display 11. Baumgarth, N. 2000. A two-phase model of B-cell activation. Immunol. Rev. conformational flexibility in CDRH3. Two different germline Abs 176: 171–180. 12. Keitel, T., A. Kramer, H. Wessner, C. Scholz, J. Schneider-Mergener, and exhibit distinct strategies to achieve polyspecificity. Collective W. Hohne. 1997. Crystallographic analysis of anti-p24 (HIV-1) monoclonal

¨ Downloaded from analysis suggests that these two mechanisms are not mutually ex- antibody cross-reactivity and polyspecificity. Cell 91: 811–820. clusive. In fact, their coexistence is essential by virtue of the re- 13. Vora, K. A., J. V. Ravetch, and T. Manser. 1997. Amplified follicular immune complex deposition in mice lacking the Fc receptor gamma-chain does not alter quirement of an infinite receptor repertoire. maturation of the B cell response. J. Immunol. 159: 2116–2124. Previously, genetic recombination of V, D, and J gene seg- 14. Wedemayer, G. J., P. A. Patten, L. H. Wang, P. G. Schultz, and R. C. Stevens. ments—alone or in conjunction with accessory processes, such 1997. Structural insights into the evolution of an antibody combining site. Sci- ence 276: 1665–1669. as nontemplated nucleotide addition and somatic hypermutation 15. Sethi, D. K., A. Agarwal, V. Manivel, K. V. Rao, and D. M. Salunke. 2006. (1, 38–40)—had been accepted as the core mechanism for rep- Differential epitope positioning within the germline antibody paratope enhances http://www.jimmunol.org/ promiscuity in the primary immune response. Immunity 24: 429–438. ertoire generation in the primary humoral response. However, 16. Maizels, N. 2005. Immunoglobulin gene diversification. Annu. Rev. Genet. 39: recent crystallographic and thermodynamic analyses of both 23–46. germline and mature Ab–Ag complexes have suggested that the 17. Notkins, A. L. 2004. Polyreactivity of antibody molecules. Trends Immunol. 25: 174–179. Ab repertoire can be further enhanced at the tertiary structural 18. Rini, J. M., U. Schulze-Gahmen, and I. A. Wilson. 1992. Structural evidence for level (7, 14, 18, 19, 34, 35). The present study provides an ele- induced fit as a mechanism for antibody-antigen recognition. Science 255: 959–965. gant correlation of CDR conformational versatility in a germline 19. Foote, J., and C. Milstein. 1994. Conformational isomerism and the diversity of antibodies. Proc. Natl. Acad. Sci. USA 91: 10370–10374. Ab with its multispecificity. Therefore, to propose a hierarchical 20. Leslie, A. G. W. 1992. Recent changes to the MOSFLM package for processing model through consolidation of the existing data for the generation film and image plate data. In Joint CCP4, Vol. 26. ESF-EAMCB Newsletter on by guest on October 2, 2021 of an almost infinite Ab repertoire is indeed attractive (Fig. 4). Protein Crystallography, p. 27–33. 21. Collaborative Computational Project, Number 4. 1994. The CCP4 suite: programs At the genetic level, independent receptors on primary B lym- for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50: 760–763. phocytes are generated through recombination of germline gene 22. Navaza, J. 1994. AMoRe: an automated package for molecular replacement. Acta Crystallogr. A 50: 157–163. segments and other accessory processes. Following genetic rear- 23. McCoy, A. J., R. W. Grosse-Kunstleve, P. D. Adams, M. D. Winn, L. C. Storoni, rangement, each resultant lymphocyte can secrete primary Abs, and R. J. Read. 2007. Phaser crystallographic software. J. Appl. Cryst. 40: 658–674. which might be capable of adopting multiple conformations, as 24. Bru¨nger, A. T. 1992. Free R value: a novel statistical quantity for assessing the accuracy of crystal structures. Nature 355: 472–475. shown in this article for BBE6.12H3 and as previously reported 25. Emsley, P, and C. Kowtan. 2004. Coot: model building tools for molecular for other germline Abs (9, 14, 41). Each conformer might be graphics. Acta Crystallogr. D Biol. Crystallogr. 60 (Part 12 Sp. Iss. 1): 2126–2132. capable of binding multiple ligands by alternative juxtapositions, 26. Chen, V. B., W. B. Arendall, III, J. J. Headd, D. A. Keedy, R. M. Immormino, G. J. Kapral, L. W. Murray, J. S. Richardson, and D. C. Richardson. 2010. as was shown in case of the germline mAb 36–65 (15). We can MolProbity: all-atom structure validation for macromolecular crystallography. envision that the collective utilization of all these mechanisms Acta Crystallogr. D Biol. Crystallogr. 66: 12–21. would effectively create an almost infinite number of receptors to 27. Kabsch, W. 1976. A solution for the best rotation to relate two sets of vectors. Acta Crystallogr. A 32: 922–923. combat unlimited Ags in the primary humoral response. 28. Henrick, E. K. 2005. Detection of protein assemblies in crystals. Computational Life Sciences 3695: 163–174. 29. Young, A. C., P. Valadon, A. Casadevall, M. D. Scharff, and J. C. Sacchettini. Acknowledgments 1997. The three-dimensional structures of a polysaccharide binding antibody to We thank Dr. Tim Manser and Dr. Kanury Rao for the gift of the anti-NP Cryptococcus neoformans and its complex with a peptide from a phage display hybridoma and other reagents; Dr. Deepak T. Nair for a critical reading library: implications for the identification of peptide mimotopes. J. Mol. Biol. 274: 622–634. of the manuscript; H.S. Sarna for technical assistance; and the BM14 30. Wilson, I. A., and R. L. Stanfield. 1994. Antibody-antigen interactions: new Beamline staff at European Synchrotron Radiation Facility for assis- structures and new conformational changes. Curr. Opin. Struct. Biol. 4: 857–867. tance during data collection. 31. Stanfield, R. L., A. Zemla, I. A. Wilson, and B. Rupp. 2006. Antibody elbow angles are influenced by their light chain class. J. Mol. Biol. 357: 1566–1574. 32. Mizutani, R., K. Miura, T. Nakayama, I. Shimada, Y. Arata, and Y. Satow. 1995. Disclosures Three-dimensional structures of the Fab fragment of murine N1G9 antibody The authors have no financial conflicts of interest. from the primary immune response and of its complex with (4-hydroxy-3- nitrophenyl)acetate. J. Mol. Biol. 254: 208–222. 33. Thorpe, I. F., and C. L. Brooks, III. 2007. Molecular evolution of affinity and flexibility in the immune system. Proc. Natl. Acad. Sci. USA 104: 8821–8826. References 34. Zimmermann, J., E. L. Oakman, I. F. Thorpe, X. Shi, P. Abbyad, C. L. Brooks, 1. Tonegawa, S. 1983. Somatic generation of antibody diversity. Nature 302: 575– III, S. G. Boxer, and F. E. Romesberg. 2006. Antibody evolution constrains 581. conformational heterogeneity by tailoring protein dynamics. Proc. Natl. Acad. 2. Weinstein, J. A., N. Jiang, R. A. White, III, D. S. Fisher, and S. R. Quake. 2009. High- Sci. USA 103: 13722–13727. throughput sequencing of the zebrafish antibody repertoire. Science 324: 807–810. 35. Yin, J., E. C. Mundorff, P. L. Yang, K. U. Wendt, D. Hanway, R. C. Stevens, and 3. Glanville, J., W. Zhai, J. Berka, D. Telman, G. Huerta, G. R. Mehta, I. Ni, P. G. Schultz. 2001. A comparative analysis of the immunological evolution of L. Mei, P. D. Sundar, G. M. Day, et al. 2009. Precise determination of the di- antibody 28B4. Biochemistry 40: 10764–10773. The Journal of Immunology 1827

36. Wedemayer, G. J., L. H. Wang, P. A. Patten, P. G. Schultz, and R. C. Stevens. 39. Lewis, S. M. 1994. The mechanism of V(D)J joining: lessons from molecular, 1997. Crystal structures of the free and liganded form of an esterolytic catalytic immunological, and comparative analyses. Adv. Immunol. 56: 27–150. antibody. J. Mol. Biol. 268: 390–400. 40. Komori, T., A. Okada, V. Stewart, and F. W. Alt. 1993. Lack of N regions in 37. Hsieh-Wilson, L. C., P. G. Schultz, and R. C. Stevens. 1996. Insights into an- antigen receptor variable region genes of TdT-deficient lymphocytes. Science tibody catalysis: structure of an oxygenation catalyst at 1.9-angstrom resolution. 261: 1171–1175. Proc. Natl. Acad. Sci. USA 93: 5363–5367. 41. Romesberg, F. E., B. Spiller, P. G. Schultz, and R. C. Stevens. 1998. Immuno- 38. Neuberger, M. S., and C. Milstein. 1995. Somatic hypermutation. Curr. Opin. logical origins of binding and catalysis in a Diels-Alderase antibody. Science Immunol. 7: 248–254. 279: 1929–1933. Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021