Adjustable Locks and Flexible Keys: Plasticity of Epitope−Paratope Interactions in Germline Antibodies

This information is current as Tarique Khan and Dinakar M. Salunke of October 2, 2021. J Immunol 2014; 192:5398-5405; Prepublished online 30 April 2014; doi: 10.4049/jimmunol.1302143 http://www.jimmunol.org/content/192/11/5398 Downloaded from

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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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Adjustable Locks and Flexible Keys: Plasticity of Epitope–Paratope Interactions in Germline Antibodies

Tarique Khan*,1 and Dinakar M. Salunke*,†

Ag recognition by independent primary Abs against a small flexible Ag with overlapping epitopes was analyzed to address the deter- minants of Ag specificity during the initial encounter. Crystal structures of two distinct dodecapeptide Ags, GDPRPSYISHLL and PPYPAWHAPGNI, in complex with the germline mAb 36-65 were determined and compared with the structures of the same Ags bound to another independent germline mAb, BBE6.12H3. For each peptide Ag, the two germline mAbs recognized overlapping epitopes, but in different topologies. The peptide structures differed, and the two paratopes attained discrete conformations, leading to different surface topologies, in a mode that can be described as adjustable locks and flexible keys. This is in contrast to mature mAbs, in which confor- mational convergence of different paratopes while binding to a common epitope in a similar conformation has been reported. These results suggest that the primary immune receptor repertoire is highly versatile as compared with its mature counterpart. Germline and mature mAbs adopt distinct mechanisms for recognizing a flexible epitope. Whereas conservation of conformational repertoire Downloaded from is a key characteristic of mature mAbs achieved through affinity maturation, the germline mAbs, at the initial stages of Ag encounter, maintain substantial plasticity, accommodating a broad specificity repertoire. The Journal of Immunology, 2014, 192: 5398–5405.

he clonal selection theory (1) predicts a correlation between recognition. Previous structural studies have provided insights on how antigenic determinants and the corresponding Abs, implying germline Ab pluripotency may enhance the BCR repertoire diversity T that each B lymphocyte expresses a unique BCR (Ab) that (4, 5, 8–10). Comparative analysis of degeneracy in interactions of http://www.jimmunol.org/ can respond to an incoming binding Ag without having been previ- germline versus affinity-matured Abs with antigenic targets would ously exposed to it. Degenerate reactivity of individual germline Abs contribute further to the understanding of the structural mechanisms has been suggested to be a physiological requirement in response to operating in the configuration of B cell immune responses. the potentially infinite antigenic repertoire to be encountered by the In the current study, we have addressed the versatility of Ag humoral immune system (2–5), although immune evasion by patho- recognition at the initial encounter by analyzing the binding of Ags gens through escape mutations provides evidence for the limitations of with overlapping epitopes by genetically independent germline the germline repertoire. In any case, the population of B cells avail- Abs. The crystal structures of mAb 36-65 in complex with the able at any point does not present the entire potential repertoire. peptide epitopes GDPRPSYISHLL (Gdp) and PPYPAWHAPGNI Therefore, to be able to respond each time exposure occurs, the hu- (Ppy) (2) were investigated by x-ray crystallography. These by guest on October 2, 2021 moral immune system has to be able to use its resources economi- structures were compared with complexes of the same peptides cally, including being able to use the same BCR to recognize different with another independent mAb BBE6.12H3 to understand the Ags as well as to use different BCRs to recognize the same Ag. structural bases of their recognition. The H chain V region of mAb Naive germline BCRs are likely to need rapid identification and 36-65 has been shown to be constructed from VH J558, DH selection upon exposure to initiate an immune response. In contrast, Fl16.1, and JH2 gene segments, whereas mAb BBE6.12H3 con- affinity-matured Abs develop through selection by prolonged exposure sists of VH 186.2, DH Fl16.1, and JH2 (11–13). Furthermore, to the dominant conformation of the Ag. It is only after the selection of mAbs 36-65 and BBE6.12H3 use L chains of the k and l isotypes, high-affinity B cells in germinal centers that affinity maturation and respectively (12, 13). L chain CDRs adopt distinct canonical clonal expansion follow (6, 7). Therefore, it is interesting to address conformations as defined by Chothia et al. (14). Thus, mAbs 36-65 the structural mechanisms adopted by germline encoded BCRs for Ag and BBE6.12H3 represent Abs of independent origin having dis- tinct CDR sequences (Fig. 1). Their structures and binding prop- *National Institute of Immunology, New Delhi 110067, India; and †Regional Centre erties have already been extensively investigated (2, 4, 5). for Biotechnology, Gurgaon 122016, India We have previously explored mechanistic details of mAb 1Current address: Department of Biochemistry, University of Texas Southwestern BBE6.12H3 binding to these peptides (4). Further analyses of the Medical Center, Dallas, TX. same peptides bound to the germline mAb 36-65 have allowed us to Received for publication August 16, 2013. Accepted for publication March 27, 2014. compare the recognition of a flexible Ag by independent germline This work was supported by the Department of Biotechnology, Government of India. Abs. Comparative analyses of these structures show that entirely The coordinates and structure factors presented in this article have been submitted to different CDR sequences are involved in the interaction with the the Research Collaboratory for Structural Bioinformatics Protein Data Bank (http:// www.pdb.org) under accession codes 4bh7 and 4bh8. peptide Ags. These data provide structural understanding of the ways in which specific recognition of a given Ag can be achieved by Address correspondence and reprint requests to Dr. Dinakar M. Salunke, Regional Centre for Biotechnology, 180, Udyog Vihar Phase-I, Gurgaon, Haryana 122016, many different VDJ/VJ combinations in the remarkably adaptable India. E-mail address: [email protected] naive immune receptor repertoire. Our studies also reveal how un- The online version of this article contains supplemental material. related Abs structurally adjust to recognize a flexible Ag and indicate Abbreviations used in this article: CNS, Crystallography and NMR System; Gdp, that the primary B cell response is likely composed of BCRs having GDPRPSYISHLL; PDB, Protein Data Bank; PEG, polyethylene glycol; Ppy, PPY- a high degree of structural adaptability. In contrast, our comparison PAWHAPGNI; RMSD, root mean square deviation. of flexible Ag recognition by germline versus affinity-matured Abs Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 shows that they adopt distinct structural mechanisms. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302143 The Journal of Immunology 5399

Materials and Methods Refinement and model building Peptides The Crystallography andNMRSystem(CNS)suitewasusedforstructure The dodecameric peptides used in this study were identified in a previous refinement (19). Both Rwork (Crystallographic R-factor) and Rfree (Free R- study by using a phage display peptide library kit (New England Biolabs, factor) (19) values were monitored during the refinement. We used 10% of the Cambridge, MA) (2). Briefly, the germline mAb BBE6.12H3 was shown total reflections in each case for calculation of Rfree values. Rigid body re- to bind to a series of independent peptides from a random phage display finement was carried out for the complete F(ab) molecule, and VH,VL,CH, library. From this set, two independent clones, 3 (GDPRPSYISHLL) and and CL domains were treated as discrete units. The models were further re- BA7-09 (PPYPAWHAPGNI), which hereafter would be referred to as Gdp fined by using positional and temperature factor refinement protocols of CNS. and Ppy, were selected for crystallographic analyses with the mAb 36-65 in COOT was used for model building and to display electron-density maps (20). the current study. These peptides were synthesized by the solid-phase method Final structures for the F(ab) were obtained after several rounds of in an automated peptide synthesizer (431A; Applied Biosystems, Foster City, manual model building in COOT (20). The peptide molecules were built CA). Peptides were cleaved from the resin by trifluoroacetic acid (Sigma- into the Ag-binding cavity of the refined F(ab) models using the electron Aldrich). A linear gradient of acetonitrile containing 0.1% trifluoroacetic density evident in the Fo-Fc maps. Water molecules were added using the acid was used to purify the peptides on a Delta Pak C18 column (Waters, water pick program in CNS. The quality of the model was checked with Milford, MA). Purified peptides were characterized by mass spectrometry. MolProbity (21). Structural models were generated using PyMOL (http:// www.pymol.org), and structural superimpositions were done using pro- Ab gram superpose in CCP4 (22). The Ab–Ag interactions and buried surface areas were analyzed using PISA (23). Structure factors and coordinates of Hybridoma cells secreting the IgG mAb 36-65 were cultured in DMEM the crystal structures of the 36-65 F(ab) bound to peptides Gdp and Ppy containing 10% FCS (12). Male BALB/c mice were g-irradiated (4 Gy) and were deposited in the Protein Data Bank(http://www.pdb.org) under the primed with Freund’s incomplete adjuvant 72 h prior to i.p. injection of accession codes 4BH8 and 4BH7, respectively. 5 3 106 hybridoma cells in 500 ml Dulbecco’s PBS per mouse. Ascitic fluid generated in the mouse peritoneal cavity was tapped after ∼4to5d.Allanimal Downloaded from experiments were approved by the Institutional Animal Ethics Committee. Results Peptide Ag Gdp and Ppy bound 36-65 F(ab) structures F(ab) preparation Sequence alignment comparing the H and L chain CDRs of mAb A three-step purification protocol was followed to purify IgG from the 36-65 and BBE6.12H3 is shown in Fig. 1. The crystal structure of ascitic fluid. The 40% ammonium sulfate–precipitated fraction containing the mAb 36-65 F(ab) in complex with the independent phage IgG was resuspended in 10 mM Tris buffer at pH 8.5. Further purification display-derived dodecapeptide Gdp was determined at 2.4 A˚ res- was carried out by affinity chromatography followed by ion-exchange http://www.jimmunol.org/ chromatography using Protein-G Sepharose and DEAE 5PW anion ex- olution (Fig. 2A). This complex was named 36-65–Gdp. The L change column on an HPLC system (Waters Delta 600; Waters), respec- and H chains were named A and B and contained 211 and 220 tively. F(ab) fragments were prepared by papain digestion of the purified residues, respectively. The structure has 98% residues in the IgG at pH 7.1. The digestion mixture was dialyzed in 10 mM Tris buffer allowed region of the Ramachandran map. The crystal structure of (pH 8) and loaded onto an anion-exchange column (DEAE 5PW) to purify the mAb 36-65 F(ab) in complex with another independent phage the F(ab) fragments. F(ab) purity was tested by SDS-PAGE, and concen- ˚ tration was estimated by the BCA protein assay kit (Pierce, Rockford, IL). display–derived dodecapeptide, Ppy, was determined at 2.9 A resolution (Fig. 2B). This complex was named 36-65–Ppy, and the Crystallization and data collection L and H chains were named A and B, respectively. In this com-

Crystals were grown by hanging-drop vapor-diffusion method (15) at 28˚C by plex, 97.5% of the residues were in the allowed region of the by guest on October 2, 2021 using a starting F(ab) concentration of 10 mg/ml. Crystals of the 36-65– Ramachandran map. In both of these complexes, the structure of peptide complex were obtained from a solution in which Gdp or Ppy peptides the entire F(ab) molecule could be built into the electron density were preincubated with F(ab) for 24 h before initiating crystallization. A 20- with the exception of a loop extending from residues Ala138B to fold molar excess of the peptide was used for cocrystallization experiments. 140B The binary complex crystallized in the presence of 15–24% polyethylene Thr . This loop region has also been shown to be disordered in glycol (PEG) of different molecular weights in 50 mM sodium cacodylate many other previously reported F(ab) structures. Crystal data and buffer in the pH range of 6.5–7 containing 0.0–0.1 M zinc chloride. Dif- refinement statistics for both the structures are shown in Table I. fraction data for the mAb 36-65 F(ab) in complex with peptides Gdp and Ppy Because the N-terminal of the peptide Gdp moves into the solvent, were collected from the crystals grown in 21% PEG 6000, 50 mM sodium cacodylate (pH 6.7), 50 mM zinc chloride, and 17% PEG 3350 in 50 mM the first three residues from the N terminus, Gly, Asp, and Pro, could sodium cacodylate (pH 6.5), respectively. Diffraction data for both complexes not be mapped. The C-terminal residue of the Gdp peptide was also were collected on the home source, RU300 (Rigaku, Tokyo, Japan). Images not visible in the electron density (Fig. 2A, Supplemental Fig. 1A). were registered on Mar345dtb image plate with an oscillation of 1˚ per im- The average temperature factors for the Gdp peptide were slightly age. The crystals were cryoprotected by soaking in mother liquor containing higher than those for the F(ab) molecule as the N-terminal residues 25% glycerol and flash frozen. Data were integrated with MOSFLM (16) and scaled with SCALA (17). were solvent exposed and did not interact with the CDR (Tables I, II). Among the bound residues, the N-terminal of the Gdp peptide is Structure determination seen to interact with the paratope generated by the 36-65 H chain The reported structure of the Ag-free mAb 36-65 F(ab) (Protein Data Bank CDRs. In the case of the 36-65–Ppy complex, the first and the last [PDB] code 2A6J) was used as an initial search model in MOLREP (18) for three residues of the Ppy peptide were not visible in the electron determining the structure of 36-65 F(ab)–peptide complexes. This model density, presumably because they were solvent exposed (Fig. 2B, produced good solutions for data corresponding to both 36-65–Gdp and 36-65–Ppy structures with correlation coefficient values of 72.2 and 67.7, Supplemental Fig. 1B). respectively. Subsequent refinements were conducted using the structures The Ab–Ag interactions were analyzed in terms of the total from these molecular replacement solutions. buried surface areas upon binding. In the case of 36-65–Gdp

FIGURE 1. Alignment of the CDR sequences of mAbs BBE6.12H3 and 36-65. Starting and ending residue numbers of each CDR are shown; identical amino acids are marked with asterisks. Deletions in CDRs are marked as a dash. 5400 GERMLINE Abs SHOW PLASTICITY OF SPECIFICITY

Table II. Interactions of Gdp peptide with mAbs 36-65 and BBE6.12H3

Gdp Residues 36-65 Residues BBE6.12H3 Residuesa Arg4 Tyr32L (4) Pro5 Tyr102B (1) Tyr32L (6), Trp91L (2) Ser6 Tyr102B (6), Gly103B (3) Tyr32L (8), Trp91L (2) Tyr7 Tyr101B (7), Tyr102B (8), Trp33H (2), Tyr97H (1) Gly103B (10) Ile8 Val 100B (1), Tyr101B (8), Tyr102B (1) Ser9 Tyr32B (3), Tyr101B (1) Trp33H (4), Arg50H (1) His10 Ser31B (4), Tyr32B (12), Arg50H (3), Asp52H (5), Gly33B (5), Ser99B (13), Ser54H (1), Gly56H (10) Val 100B (13),Tyr101B (10), Tyr106B (5) Leu11 Tyr50B (3), Asn52B (5), Tyr101B (1), Tyr106B (5) Epitope–paratope interactions were calculated using van der Waals radii in con- tact program of CCP4 (16). The 12-mer peptide (Gdp) residues, which were common in both complexes, are shown in boldfaced text. The number of interactions shown by each Ab residue is shown in parentheses. aData taken from Khan and Salunke (4). Downloaded from

Cross-reacting peptide Ag: Gdp in different germline Ab environments Comparison of the structure of Gdp determined in this study in complex with mAb 36-65 with that in the previously reported http://www.jimmunol.org/ FIGURE 2. Two independent crystal structures of the germline mAb complex with mAb BBE6.12H3 (PDB code 2Y06) (4), provided 36-65 F(ab) bound to peptide Ags Gdp (A) and Ppy (B). The two peptide- interesting insights into the structural behavior of Gdp in the bound states are shown as ribbons highlighting secondary structural fea- context of BCR recognition. As shown in Fig. 3A, the paratope tures. Peptide molecules are shown as blue sticks. Surface representations surfaces of mAbs 36-65 and BBE6.12H3 bound to Gdp were very of mAb 36-65 in complex with Gdp and Ppy peptides, respectively, are shown in the bottom panel. The interacting regions of the paratope surfaces different. Superimpositions of these complexes show that the CDR are highlighted in red along with the interacting residues (sticks). conformations of these two germline mAbs differ substantially. Although CDRs H1 and H2 seem to be similar (root mean square complex, the total buried surface areas of F(ab) and peptide were deviation [RMSD] ,0.5 A˚ ), as they adopt the same canonical by guest on October 2, 2021 2180 and 504 A˚ 2, respectively. In the case of 36-65–Ppy complex, conformations. Although the sequences of CDRH1 and CDRH2 the corresponding total buried surface areas were 2290 and 426 A˚ 2, were also similar (Fig. 1), the individual interacting residues in- respectively. volved were very different. The elbow angles and the buried surface areas of the two F(ab) molecules differ by 10˚ and 670 A˚ 2, Table I. Data collection and refinement statistics respectively. Eight residues from Arg4P to Leu11P could be unambiguously 36-65–Gdp 36-65–Ppy traced into the electron density in the 2Fo-Fc map for the 36-65– Gdp complex (Supplemental Fig. 1A). In contrast, nine residues Data-processing statistics 2P 10P Space group P21 P21 from Asp to His were evident in the BBE6.12H3–Gdp com- Cell dimensions plex. Structural comparison of Gdp in the two Ab complexes a, b, c (A˚ ) 54.4,77.0, 59.2 54.5, 76.8, 59.1 revealed that the gross topology of the peptide is conserved. They ˚ b ( ) 101.7 102.3 both exhibit approximately a right-angled arrangement of the Maximum resolution (A˚ ) 58–2.4 57–2.9 backbone with a change in direction at the proline residue (Fig. 3B). Rmerge (%) 10.0 (40.0) 12.0 (32.0) Average (I)/(Sig I) 12.0 (3.2) 9.4 (3.9) However, neither the backbone nor the side chain atoms of any Completeness (%) 100.0 (100.0) 97.8 (99.7) residue were superimposable. Comparison of RMSD values for each No. of unique reflections 19,076 10,548 peptide residue in terms of the Ca positions (2.5 A˚ )aswellasthe Multiplicity 4.5 (4.5) 4.7 (4.7) ˚ Refinement statistics side-chain atoms (4.6 A) also indicated that the conformations of the Resolution range (A˚ ) 50–2.4 57.8–2.89 individual residues of the Gdp peptide were different when bound to No. of reflections 18,818 9,490 mAbs 36-65 and BBE6.12H3. Interestingly, in both complexes, the Rwork/Rfree (%) 23.0/25.1 22.9/25.4 peptide bound in the same orientation, albeit in different regions of No. of atoms the paratope (Fig. 4A, 4B). The backbone and the side chains of the Protein 3,310 3,313 Peptide 69 68 peptide residues oriented differently to interact with distinct para- Water 190 77 tope residues in the 36-65–Gdp and BBE6.12H3–Gdp complexes B-factor (Fig. 4A, 4B). Some peptide residues interacted with similar amino Protein 24.5 22.5 acids but their spatial locations on the paratope differed substan- Peptide 84.4 64.5 Water 32.5 23.0 tially, as outlined in Tables II and III and Fig. 3C and 3D. RMSDs The middle region of the peptide (i.e., the residues PSYISH) was Bond length (A˚ ) 0.007 0.01 conformationally similar when bound to either mAb 36-65 or mAb Bond angle (˚) 1.7 1.8 BBE6.12H3. The backbone conformations of this region were Data in parentheses are for highest-resolution shell. similar, although side-chain orientations differed. This is particularly The Journal of Immunology 5401

FIGURE 3. Differences in the CDR conformations and the interactional network of mAbs, 36-65, and BBE6.12H3 to recognize the peptide Ag Gdp. (A) Stereoscopic view of the structural alignment of the CDRs of the Gdp peptide bound mAbs 36-65 (blue) and BBE6.12H3 (orange). (B) Structure alignment to compare the conformations of the peptide Gdp when bound to mAbs 36-65 (red) and Downloaded from BBE6.12H3 (green). Stereoscopic diagrams displaying interacting residues (within a 4-A˚ distance) of the bound peptide are shown as thin sticks, and the stretch of the Gdp peptide evident in the electron-density map of its com- plexes with mAbs 36-65 and BBE6.12H3 is shown as thick sticks. (C) 36-65–Gdp. (D) BBE6.12H3–Gdp. http://www.jimmunol.org/ by guest on October 2, 2021

evident for Tyr7P and His10P (Fig. 3B). In addition, the CDR these residues, were entirely different (Table II). One residue of residues of mAbs BBE6.12H3 and 36-65, which interact with the Gdp peptide, Ile8P, was solvent exposed in the BBE6.12H3– Gdp complex, but the same residue had numerous interactions in 36-65–Gdp (Tables II, III).

Table III. Comparison of atomwise polar interactions of 36-65 and BBE6.12H3 with Gdp peptide

Gdp [Atoms] 36-65 [Atoms] BBE6.12H3 [Atoms]a Arg4 [N] L:Tyr32 [OH] Pro5 [N] L:Tyr32 [OH] Ser6 [N] B:Tyr102 [OH] L: Trp91 [NE1] Ser6 [O] B:Gly103 [N] L:Trp91 [NE1] Ser6 [OG] H:Tyr95 [OH] Tyr7 [O] B:Tyr102 [N], Gly103 [N] Ile8 [N] B: Tyr101 [O] Ile8 [O] B: Tyr101 [N] Ser9 [O] H:Arg50 [NH2] Ser9 [N] B:Tyr101 [O] Ser9 [OG] B:Tyr32 [OH] His10 [N] B:Tyr101 [N] FIGURE 4. Comparison of Gdp peptide-binding mode on independent Ab His10 [O] B: Ser31 [O] H: Arg50 [NE], paratope surfaces. Peptide is displayed as sticks. In the surface view, mAbs 36-65 Arg50 [NH2] andBBE6.12H3areshowninblueandmagenta, respectively. The interacting His10 [ND1] B:Ser31 [O], Ser99 [O] 10 33 99 paratope surfaces in both complexes are colored orange. Close-up of the inter- His [NE2] B:Gly [N], Ser [O] acting Connolly surfaces of the Abs are decorated according to their hydropathy Polar contacts were calculated using PISA (23). feature (red, hydrophobic; blue, charged). (A) 36-65–Gdp. (B) BBE6.12H3–Gdp. aData taken from Khan and Salunke (4). 5402 GERMLINE Abs SHOW PLASTICITY OF SPECIFICITY

The Gdp peptide bound at different sites in the Ag-combining the case of BBE6.12H3-Gdp, both L and H chain CDRs contributed grooves of mAbs BBE6.12H3 and 36-65, involving entirely dif- equally to the polar interactions. Detailed comparisons of atomwise ferent residues (Fig. 3C, 3D). Gdp did not interact with the L chain polar contacts of each residue are provided in Table III. CDRs in the 36-65–Gdp complex. Even in the H chain CDRs, entirely different sets of residues were involved in the interaction Cross-reacting peptide Ag: Ppy in different germline Ab with the two mAbs (Fig. 3C, 3D, Table II). In BBE6.12H3–Gdp, environments several residues from the L and H chains made van der Waals The structure of Ppy bound to the mAb BBE6.12H3 (PDB code contacts with the peptide residues (Table II). In the 36-65–Gdp 2Y07) has been reported previously (4). A comparative analysis of complex, the peptide Ag bound over the CDRH3 loop in a zigzag the 36-65–Ppy and BBE6.12H3-Ppy paratopes revealed distinct pattern. Thus, the majority of the van der Waals contacts were made topologies and charge distributions (Figs. 5A, 6A, 6B). Super- by CDRH3 residues in 36-65–Gdp (Fig. 3C, Table II). In 36-65– imposition of the secondary structural elements also showed dif- Gdp, Tyr7P and Ile8P were oriented toward the CDRH3 and hence ferent CDR conformations except for CDRs H1 and H2 (Fig. 5A). had additional interactions when compared with those in mAb Analyses at the level of individual interacting residues indicated BBE6.12H3. Similarly, His10P in the 36-65–Gdp complex made entirely different side-chain orientations in all of the CDRs. more contacts as it snugly fit into a groove between CDRH3 and The elbow angles and the buried surface areas differed by 10˚ and CDRH1 (Fig. 4A, 4B) and also contributed maximally toward the 220 A˚ 2, respectively. buried surface area. The majority of the polar interactions in 36-65– The structures of the Ppy peptide bound to the germline mAbs Gdp were formed by the H chain CDRs H1 and H3. In contrast, in 36-65 and BBE6.12H3 were superimposed to compare peptide Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021 FIGURE 5. The differences in the CDR conformations and the interactional network of mAbs 36-65 and BBE6.12H3 to recognize the peptide Ag Ppy. (A) Stereo- scopic view of the structural alignment of the CDRs of the Ppy peptide-bound mAbs 36-65 (blue) and BBE6.12H3 (orange). (B) Structure alignment to compare the con- formations of the peptide Ppy when bound to mAbs 36-65 (green) and BBE6.12H3 (red). Residues involved in the b-turn formation in Ppy peptide are marked with dotted square (green) and dotted circle (red) in 36-65–Ppy and BBE6.12H3–Ppy, respectively. Stereoscopic views display- ing interacting residues (within a 4-A˚ distance) of the bound peptide are shown in thin-stick and the stretch of the Gdp peptide evident in the electron density map of its complexes with mAbs 36-65 and BBE6.12H3 is shown in thick-stick representation. (C) 36-65–Ppy. (D) BBE6.12H3–Ppy. The Journal of Immunology 5403

Table IV. Interactions of Ppy peptide with mAbs 36-65 and BBE6.12H3

Ppy Residuesa 36-65 Residues BBE6.12H3 Residuesa Pro2 Trp91L (4), Ser93L (2), Asn94L (2) Tyr3 Lys59B (5), Lys65B (1) Trp91L (6), Trp33H (7), Arg50H (8), Ala58H (2) Pro4 Tyr57B (4), Lys59B (3) Trp33H (2), Arg50H (1) Ala5 Tyr57B (3) Trp6 Tyr50B (1), Tyr106B (3) Trp33H (8), Tyr95H (3), Tyr97H (2) His7 Tyr32A (8), Gly91A (4), Asn92A (5), Arg96A (3), Gly104B (7), Ser105B (8) Ala8 Asn92A (3), Thr93A (4), Leu94A (6), Arg96A (1) Pro9 Thr93A (4), Leu94A (7) Gly10 Leu94A (2) FIGURE 6. Comparison of Ppy peptide-binding mode on independent Epitope–paratope interactions were calculated using van der Waals radii in con- Ab paratope surfaces. Peptide is displayed as sticks. In the surface view,

tact program of CCP4 suite (16). The 12-mer peptide (Ppy) residues, which were Downloaded from mAbs 36-65 and BBE6.12H3 are shown in blue and magenta, respectively. common in both complexes, are shown in boldfaced text. The number of interactions The interacting paratope surfaces in both complexes are colored orange. shown by each Ab residue is shown in parentheses. a Close-up of the interacting Connolly surfaces of the Abs are decorated Data taken from Khan and Salunke (4). according to their hydropathy feature (red, hydrophobic; blue, charged). (A) 36-65–Ppy. (B) BBE6.12H3–Ppy. conformation and interactions of these residues were different in the two Ab environments (Fig. 5B, Table IV). In the 36-65–Ppy conformation in the two complexes (Fig. 5B). Interestingly, Ppy complex, Pro2P did not interact with the paratope, as it was solvent http://www.jimmunol.org/ adopted a b-turn conformation in both of the structures. However, exposed. In contrast, the same residue made several contacts with this turn consisted of distinct sets of residues: AWHAP and YPAW the paratope in the BBE6.12H3–Ppy complex (Table IV). These in 36-65–Ppy and BBE6.12H3–Ppy, respectively (Fig. 5B). Su- observations demonstrate that these two germline mAbs use dif- perimposition of the Ppy peptide from the two complexes revealed ferent kinds of interactions and with different sets of residues for different conformations. The RMSD values in the positions of Ca recognizing the same peptide epitope. and side chains were 2.1 and 4.6 A˚ , respectively. The peptide residues had considerable differences in their side-chain ori- Discussion entations, perhaps matching the surface charge distribution on the

The phenomenon of degeneracy in Ag binding in germline mAbs is by guest on October 2, 2021 paratope surfaces of mAbs 36-65 and BBE6.12H3 (Fig. 5A, 5B). likely to be significant for enabling responsiveness to a diversity of 3P 6P Changes in the side chains of Tyr and Trp were particularly pathogens (2, 5, 8, 10). The high specificity of recognition in the 2P 9P clearly evident. Although eight residues from Pro to Pro could immune system is countered by evasion strategies in pathogens be traced into the electron density in the 2Fo-Fc map for the which, among other modalities (24), use mechanisms to change 1P 6P 36-65–Ppy complex, only six residues from Pro to Trp were their antigenic surfaces (25–27) and/or mimic host moieties (28, discernible in the BBE6.12H3–Ppy complex (Fig. 5C, 5D). The 29). Structurally ill-defined antigenic determinants could well add electron-density map for the Ppy peptide is shown in Supplemental further difficulties for the host during the initial encounter when Fig. 1B. Although detailed conformational features of the peptide germline Abs would be involved. in the two structures were distinct, the overall fold of the peptide It was evident that the two germline mAbs used in this study, remained similar (Fig. 5B). BBE6.12H3 and 36-65, adopt different structural strategies while The residues of the two independent F(ab) molecules involved in recognizing a common epitope, even though their affinities are interactions with Ppy peptide are shown in Table IV and Fig. 5C comparable. The Ag-combining sites in the two mAbs show overlap, and 5D. As is evident in Table V, polar interactions were observed with similar footprints for the common epitope. However, they adopt in both complexes but with different residues of the mAbs 36-65 entirely different paratope topologies with substantial differences in and BBE6.12H3. In the 36-65–Ppy complex, whereas the majority of the van der Waals contacts were formed by CDRL3, some residues from CDRL1, CDRH2, and CDRH3 also contributed to it Table V. Comparison of atomwise polar interactions of 36-65 and (Fig. 5C, 5D, Table IV). The peptide Ppy bound at different sites BBE6.12H3 with Ppy peptide in the mAbs 36-65 and BBE6.12H3. The middle region of the 57B peptide made van der Waals contacts with Tyr . The C terminus Ppy [Atoms] 36-65 [Atoms] BBE6.12H3 [Atoms]a of the peptide Ag showed a major interaction with CDRL3 Pro2 [N] L:Tyr32 [OH] (Fig. 6A, 6B). The majority of the van der Waals contacts in this Tyr3 [OH] B:Lys59 [NZ], Lys65 [NZ] H:Arg50 [NE], case were through polar residues. In contrast, van der Waals 50Arg50 [NH1] contacts in BBE6.12H3-Ppy complex were formed with all types Pro4 [O] B:Tyr57 [OH], Lys59 [NZ] Ala5 [N] B:Tyr57 [OH] of amino acids. The N terminus of the peptide in the complexes 7 96 with mAbs 36-65 and BBE6.12H3 bound at distinct paratope His [N] B:Arg [NH2] His7 [ND1] B:Gly104 [O], Ser105 [N] surfaces because it had major interactions with CDRH2 and CDRL3, His7 [NE2] A:Asn92 [O] respectively (Fig. 5C, 5D, Table IV). Pro9 [O] A:Leu94 [N] Out of the 12 peptide residues, 5 comprising the sequence Polar contacts were calculated using PISA (23). PYPAW were seen bound to both of the mAbs. However, the aData taken from Khan and Salunke (4). 5404 GERMLINE Abs SHOW PLASTICITY OF SPECIFICITY the conformations of the CDR loops. The common epitopes also to further delineate the structural mechanisms involved and their adopt distinct conformations when bound to different mAbs. physiological significance. However, greater conformational rearrangements were observed in the germline mAb paratopes than in the corresponding epitopes. Acknowledgments These findings indicate that structural plasticity mechanisms to We thank Drs. Tim Manser and K.V.S. Rao for the gift of the hybridomas, recognize the Ag are inherent to the germline mAbs. Drs. Deepak T. Nair, Jasmita Gill, and Satyajit Rath for critically reading Interestingly, in previous work, independent mature Ab clones the manuscript, and H.S. Sarna for technical assistance. exhibited structural convergence against an immunodominant epitope, PS1, known to be flexible in solution (30). The PS1 epitope Disclosures was driven to a common conformational state and the CDRs of each The authors have no financial conflicts of interest. of the independent mature mAbs against this epitope adopted identical conformations while binding to it (31). Thus, contrary to the broad conformational repertoire of the primary Abs observed in References the current study, that of the mature Abs was conserved during Ag 1. Burnet, F. M. 1957. A modification of Jerne’s theory of antibody production using the concept of clonal selection. Aust. J. Sci. 20: 67–69. binding (31–34). 2. Manivel, V., F. Bayiroglu, Z. Siddiqui, D. M. Salunke, and K. V. Rao. 2002. The In this context, the mode of physical contacts between an Ag primary antibody repertoire represents a linked network of degenerate antigen and two distinct germline mAbs as deciphered in the current study specificities. J. Immunol. 169: 888–897. 3. Nguyen, H. P., N. O. Seto, C. R. MacKenzie, L. Brade, P. Kosma, H. Brade, and might have implications for rapid recognition of flexible epitopes by S. V. Evans. 2003. Germline antibody recognition of distinct carbohydrate a limited Ab repertoire. It is plausible that the enhanced flexibility epitopes. Nat. Struct. Biol. 10: 1019–1025. in CDRs compared with that in the peptide Ags might enable re- 4. Khan, T., and D. M. Salunke. 2012. Structural elucidation of the mechanistic Downloaded from basis of degeneracy in the primary humoral response. J. Immunol. 188: 1819– cruitment of multiple independent naive B cell clones. It has been 1827. shown that both the framework region and the CDRs have a con- 5. Sethi, D. K., A. Agarwal, V. Manivel, K. V. Rao, and D. M. Salunke. 2006. Differential epitope positioning within the germline antibody paratope enhances siderable amount of inherent conformational plasticity (35–37). promiscuity in the primary immune response. Immunity 24: 429–438. Therefore, it is not surprising that distinct germline Abs recognize 6. Ochiai, K., M. Maienschein-Cline, M. Mandal, J. R. Triggs, E. Bertolino, the same epitope by rearranging the CDR conformations. This may R. Sciammas, A. R. Dinner, M. R. Clark, and H. Singh. 2012. A self-reinforcing

regulatory network triggered by limiting IL-7 activates pre-BCR signaling and http://www.jimmunol.org/ well have implications of Ag specificity beyond the naive BCR differentiation. Nat. Immunol. 13: 300–307. repertoire, because Kaji et al. (38) have shown in a recent report that 7. Berek, C., and C. Milstein. 1987. Mutation drift and repertoire shift in the the B cell memory can contain both germline-encoded and so- maturation of the immune response. Immunol. Rev. 96: 23–41. 8. James, L. C., P. Roversi, and D. S. Tawfik. 2003. Antibody multispecificity matically mutated BCRs. mediated by conformational diversity. Science 299: 1362–1367. Polyclonal Abs generated against an Ag are generally expected 9. Notkins, A. L. 2004. Polyreactivity of antibody molecules. Trends Immunol. 25: 174–179. to bind diverse epitopes with distinct structures. If the Ag is small, 10. Wedemayer, G. J., P. A. Patten, L. H. Wang, P. G. Schultz, and R. C. Stevens. such as a dodecapeptide as in the present case, the polyclonal 1997. Structural insights into the evolution of an antibody combining site. Sci- response might become effectively monospecific due to the in- ence 276: 1665–1669. 11. Wong, Y. W., D. S. Gill, B. Parhami-Seren, M. K. Short, S. R. Sompuram, and evitable overlap of reacting surfaces. If this small Ag is flexible,

M. N. Margolies. 1998. Structural requirements for a specificity switch and for by guest on October 2, 2021 different Ab clones binding to it may possibly recognize different maintenance of affinity using mutational analysis of a phage-displayed anti- topological states of the molecule. Indeed, the peptide Ags Gdp and arsonate antibody of Fab heavy chain first complementarity-determining re- gion. J. Immunol. 160: 5990–5997. Ppy binding to two independent mAbs BBE6.12H3 and 36-65 in 12. Vora, K. A., J. V. Ravetch, and T. Manser. 1997. Amplified follicular immune distinct topological states reflects this possibility. complex deposition in mice lacking the Fc receptor gamma-chain does not alter maturation of the B cell response. J. Immunol. 159: 2116–2124. The inherent conformational potential of a flexible peptide is 13. Wong, Y. W., P. H. Kussie, B. Parhami-Seren, and M. N. Margolies. 1995. of considerable structural interest. Functionally relevant conforma- Modulation of antibody affinity by an engineered amino acid substitution. J. tional preferences of flexible molecules with known physiological Immunol. 154: 3351–3358. 14. Chothia, C., A. M. Lesk, A. Tramontano, M. Levitt, S. J. Smith-Gill, G. Air, receptors can indeed be deciphered in the form of their complexes. S. Sheriff, E. A. Padlan, D. Davies, W. R. Tulip, et al. 1989. Conformations of However, the conformational preferences of linear peptides, which immunoglobulin hypervariable regions. Nature 342: 877–883. do not embody a well-defined structural motif and do not have 15. Wlodawer, A., and K. O. Hodgson. 1975. Crystallization and crystal data of monellin. Proc. Natl. Acad. Sci. USA 72: 398–399. known physiological receptors, are harder to dissect. Abs as 16. Leslie, A. G. W. 1992. Joint CCP4 and ESFEACMB newsletter on protein. receptors, particularly when they have flexible binding sites, as is Protein Crystallogr. 26: 27–33. 17. Collaborative Computational Project, Number 4. 1994. The CCP4 suite: pro- the case in germline Abs, can provide snapshots of possible con- grams for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50: formational preferences for such flexible peptides. This is particu- 760–763. larly attractive when structural complexes of more than one such 18. Navaza, J. 1994. AMoRe: an automated package for molecular replacement. Acta Crystallogr. A 50: 157–163. independent flexible receptor with a common ligand are available. 19. Brunger,€ A. T. 1992. Free R value: a novel statistical quantity for assessing the The crystal structures of the two linear peptides bound to two dif- accuracy of crystal structures. Nature 355: 472–475. ferent germline Abs determined in the current study provided this 20. Emsley, P., and K. Cowtan. 2004. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60: 2126–2132. scenario. 21. Chen, V. B., W. B. Arendall, III, J. J. Headd, D. A. Keedy, R. M. Immormino, Our data illustrate the structural versatility of germline Abs, G. J. Kapral, L. W. Murray, J. S. Richardson, and D. C. Richardson. 2010. MolProbity: all-atom structure validation for macromolecular crystallography. which is relevant during initial antigenic encounter, whereas ma- Acta Crystallogr. D Biol. Crystallogr. 66: 12–21. ture Abs of the secondary immune response do not appear to retain 22. Kabsch, W. 1976. A solution for the best rotation to relate two sets of vectors. such a wide conformational diversity. Indeed, the many instances of Acta Crystallogr. A 32: 922–923. 23. Krissinel, E., and K. Henrick. 2005. Detection of protein assemblies in crystals. independent mature Abs showing convergence of their paratope Comput. Life Sci. 3695: 163–174. topologies while binding a common epitope support this possibility 24. Mulhern, O., B. Harrington, and A. G. Bowie. 2009. Modulation of innate im- (33, 39–41). Such use of distinct structural strategies by BCRs at mune signalling pathways by viral proteins. Adv. Exp. Med. Biol. 666: 49–63. 25. Manel, N., and D. R. Littman. 2011. Hiding in plain sight: how HIV evades germline versus mature stages may have physiological significance. innate immune responses. Cell 147: 271–274. Because the current analysis is based on 12-mer peptides and their 26. Sandbulte, M. R., K. B. Westgeest, J. Gao, X. Xu, A. I. Klimov, C. A. Russell, D. F. Burke, D. J. Smith, R. A. Fouchier, and M. C. Eichelberger. 2011. Dis- interactions with germline mAbs, additional studies with protein cordant antigenic drift of neuraminidase and hemagglutinin in H1N1 and H3N2 Ags and their cognate Abs, both germline and mutated, would help influenza viruses. Proc. Natl. Acad. Sci. USA 108: 20748–20753. The Journal of Immunology 5405

27. Hilleman, M. R. 2004. Strategies and mechanisms for host and pathogen survival 17/9 and three different Fab-peptide complexes. J. Mol. Biol. 234: 1098– in acute and persistent viral infections. Proc. Natl. Acad. Sci. USA 101(Suppl. 2): 1118. 14560–14566. 36. Stanfield, R. L., M. Takimoto-Kamimura, J. M. Rini, A. T. Profy, and 28. Moran, A. P., M. M. Prendergast, and B. J. Appelmelk. 1996. Molecular mimicry I. A. Wilson. 1993. Major antigen-induced domain rearrangements in an anti- of host structures by bacterial lipopolysaccharides and its contribution to disease. body. Structure 1: 83–93. FEMS Immunol. Med. Microbiol. 16: 105–115. 37. Arevalo, J. H., C. A. Hassig, E. A. Stura, M. J. Sims, M. J. Taussig, and 29. Stebbins, C. E., and J. E. Gala´n. 2001. Structural mimicry in bacterial virulence. I. A. Wilson. 1994. Structural analysis of antibody specificity. Detailed com- Nature 412: 701–705. parison of five Fab’-steroid complexes. J. Mol. Biol. 241: 663–690. 30. Nayak, B. P., R. Tuteja, V. Manivel, R. P. Roy, R. A. Vishwakarma, and 38. Kaji, T., A. Ishige, M. Hikida, J. Taka, A. Hijikata, M. Kubo, T. Nagashima, K. V. Rao. 1998. B cell responses to a peptide epitope. V. Kinetic regulation of Y. Takahashi, T. Kurosaki, M. Okada, et al. 2012. Distinct cellular pathways repertoire discrimination and antibody optimization for epitope. J. Immunol. select germline-encoded and somatically mutated antibodies into immunological 161: 3510–3519. memory. J. Exp. Med. 209: 2079–2097. 31. Nair, D. T., K. Singh, Z. Siddiqui, B. P. Nayak, K. V. Rao, and D. M. Salunke. 39. Xu, Y., P. A. Ramsland, J. M. Davies, M. Scealy, K. S. Nandakumar, 2002. Epitope recognition by diverse antibodies suggests conformational con- R. Holmdahl, and M. J. Rowley. 2004. Two monoclonal antibodies to precisely vergence in an antibody response. J. Immunol. 168: 2371–2382. the same epitope of type II collagen select non-crossreactive phage clones by 32. Goldman, E. R., W. Dall’Acqua, B. C. Braden, and R. A. Mariuzza. 1997. phage display: implications for autoimmunity and molecular mimicry. Mol. Analysis of binding interactions in an idiotope-antiidiotope protein-protein Immunol. 41: 411–419. complex by double mutant cycles. Biochemistry 36: 49–56. 40.Ramsland,P.A.,W.Farrugia,T.M.Bradford,P.MarkHogarth,and 33. Berger, C., S. Weber-Bornhauser, J. Eggenberger, J. Hanes, A. Pluckthun,€ and A. M. Scott. 2004. Structural convergence of antibody binding of carbo- H. R. Bosshard. 1999. Antigen recognition by conformational selection. FEBS hydrate determinants in Lewis Y tumor antigens. J. Mol. Biol. 340: 809– Lett. 450: 149–153. 818. 34. Fields, B. A., F. A. Goldbaum, X. Ysern, R. J. Poljak, and R. A. Mariuzza. 1995. 41. Scheid, J. F., H. Mouquet, B. Ueberheide, R. Diskin, F. Klein, T. Y. Oliveira, Molecular basis of antigen mimicry by an anti-idiotope. Nature 374: 739–742. J. Pietzsch, D. Fenyo, A. Abadir, K. Velinzon, et al. 2011. Sequence and 35. Schulze-Gahmen, U., J. M. Rini, and I. A. Wilson. 1993. Detailed analysis structural convergence of broad and potent HIV antibodies that mimic CD4 of the free and bound conformations of an antibody. X-ray structures of Fab binding. Science 333: 1633–1637. Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021