Structural Evaluation of a Mimicry-Recognizing Paratope: Plasticity in Antigen−Antibody Interactions Manifests in Molecular Mimicry This information is current as of September 28, 2021. Suman Tapryal, Vineet Gaur, Kanwal J. Kaur and Dinakar M. Salunke J Immunol published online 3 June 2013 http://www.jimmunol.org/content/early/2013/06/01/jimmun ol.1203260 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 © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published June 3, 2013, doi:10.4049/jimmunol.1203260 The Journal of Immunology

Structural Evaluation of a Mimicry-Recognizing Paratope: Plasticity in Antigen–Antibody Interactions Manifests in Molecular Mimicry

Suman Tapryal,*,1 Vineet Gaur,*,1 Kanwal J. Kaur,* and Dinakar M. Salunke*,†

Molecular mimicry manifests antagonistically with respect to the specificity of immune recognition. However, it often occurs because different Ags share surface topologies in terms of shape or chemical nature. It also occurs when a flexible paratope accommodates dissimilar Ags by adjusting structural features according to the antigenic epitopes or differential positioning in the Ag combining site. Toward deciphering the structural basis of molecular mimicry, mAb 2D10 was isolated from a maturing immune response elicited against methyl a-D-mannopyranoside and also bound equivalently to a dodecapeptide. The physicochemical evidence of this carbohydrate– peptide mimicry in the case of mAb 2D10 had been established earlier. These studies had strongly suggested direct involvement of Downloaded from a flexible paratope in the observed mimicry. Surprisingly, comparison of the Ag-free structure of single-chain variable fragment 2D10 with those bound to sugar and peptide Ags revealed a conformationally invariant state of the Ab while binding to chemically and structurally disparate Ags. This equivalent binding of the two dissimilar Ags was through mutually independent interactions, dem- onstrating functional equivalence in the absence of structural correlation. Thus, existence of a multispecific, mature Ab in the secondary immune response was evident, as was the plasticity in the interactions while accommodating topologically diverse Ags. Although our data highlight the structural basis of receptor multispecificity, they also illustrate mechanisms adopted by the immune http://www.jimmunol.org/ system to neutralize the escape mutants generated during pathogenic insult. The Journal of Immunology, 2013, 191: 000–000.

pecific molecular recognition involves receptor and ligand mimicry has also been exploited as a conceptual tool for rational surfaces to complement each other in terms of shape and drug design to identify inhibitors as the substrate mimics of var- S charge to achieve physiologically meaningful interactions. It ious therapeutic target proteins and enzymes (15, 16). is not uncommon, however, that completely unrelated molecules Methyl a-D-mannopyranoside and a dodecapeptide (DVFYPY- share common receptors, possibly through similar structural and/or PYASGS) known to bind Con A with comparable affinities have chemical features involved in recognition and binding, resulting in been studied extensively as a model system to address molecular

molecular mimicry (1–3). Molecular mimicry often occurs by de- mimicry in the humoral immune response (17–19). Polyclonal sera by guest on September 28, 2021 sign and manifests as a control during various regulatory mecha- generated against methyl a-D-mannopyranoside cross-recognize the nisms (4). It also occurs as an accidental encounter of structural dodecapeptide, and correspondingly antipeptide sera cross-react resemblances, particularly in the immune system, sometimes with the mannopyranoside (20). Additionally, dodecapeptide was culminating in pathological conditions such as autoimmune dis- observed to cross-boost the antisugar response in mice (21). The orders (5–8). The molecular mimicry between self and viral or structure of dodecapeptide in complex with Con A was determined. bacterial peptides generally leads to the activation of self-reactive Comparison of sugar and peptide structures bound to Con A T cells resulting in autoimmune pathologies, for example, multi- revealed that although peptide displayed structural and functional ple sclerosis, autoimmune hepatitis, and myocarditis (9–11). The mimicry to sugar, it did not bind at the carbohydrate binding site of humoral immune system has also been shown to produce auto- Con A, but adjacent to it (22, 23). Thus, although these data sug- reactive Abs, some of which are also associated with cancers (12, gested functional quasi-equivalence between the dodecapeptide and 13). In case of the anti–U1-70 kDa autoimmunity, the host protein the carbohydrate moiety, the precise topological correlation be- shares T cell epitopes with 13 fungal proteins (14). Molecular tween the two molecules was elusive. Although the mimicry between the sugar and peptide molecules was functionally elucidated during the onset as well as the *National Institute of Immunology, New Delhi 110067, India; and †Regional Centre progression of the humoral immune response, further analyses at for Biotechnology, Gurgaon 122016, India the molecular level were considered best addressed using rel- 1 S.T. and V.G. contributed equally to this work. evant mAbs. Indeed, the mAb 2D10 generated against the man- Received for publication November 27, 2012. Accepted for publication May 3, 2013. nopyranoside moiety bound both the methyl a-D-mannopyranoside This work was supported by a grant from the Department of Biotechnology, Gov- and the dodecapeptide (DVFYPYPYASGS) with equivalent af- ernment of India and by a J.C. Bose fellowship. finity (24), providing an elegant model for structural and func- The sequences presented in this article have been submitted to the Research Collaboratory tional investigations. Thermodynamic and in silico studies had for Structural Bioinformatics Protein Data Bank (http://www.rcsb.org) under accession codes 4H0G, 4H0I, and 4H0H. suggested a flexible Ag combining site as a basis for the promis- Address correspondence and reprint requests to Dr. Dinakar M. Salunke, Regional cuous recognition of these otherwise chemically distinct molecules Centre for Biotechnology, 180 Udyog Vihar Phase I, Gurgaon 122016, India. E-mail (25). Because 2D10-Fab could not be crystallized, a shorter re- address: [email protected] combinant single-chain variable fragment (scFv; single-chain Ab) Abbreviations used in this article: rmsd, root-mean-square deviation; scFv, single- molecule 2D10 was constructed that facilitated application of crys- chain variable fragment. tallographic methods to understand functional mimicry involving the Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 mannopyranoside and the dodecapeptide (26).

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203260 2 PLASTICITY IN Ag–Ab INTERACTIONS

In this study we present crystal structures of scFv of the mAb used for model building. Throughout the procedure, Rwork and Rfree (30) 2D10 in an Ag-free state as well as in complex with the sugar, methyl were used to monitor the progress of the refinement by omitting 10% of the data, chosen randomly, for calculation of R values. Once the model for a-D-mannopyranoside, and the dodecapeptide. Superimposition of free the scFv 2D10 molecules had been completed, density visible at the Ag these structures revealed that the CDRs of the Ab had a predefined binding site could be interpreted as methyl a-D-mannopyranoside mole- invariant conformation in all the three forms, implying that the Ag cule in case of the scFv–sugar complex. In the case of the scFv–peptide combining site may have a rigid structure. Analysis of the inter- complex, connected density at the Ag combining site allowed residues P3– actions between the paratope and the respective Ags revealed a P9 of the peptide to be fitted in electron density map. The model of peptide was fitted by visualizing 2Fo-Fc map at a s cutoff of 0.7 and Fo-Fc map at unique mechanism for the manifestation of carbohydrate–peptide a s cutoff of 1.6. The quality of the model was controlled with MolProbity mimicry with respect to their recognition by the immune receptors. (32). Superimpositions of the entire proteins and their various submolecular components were carried out using superpose (33) from the CCP4 program suite as well as from PyMol. The Ag and Ab interface interactions were Materials and Methods determined using Contact (CCP4 program suite) and PISA (34). The epitope– Ag molecules paratope interactions and the buried surface areas for each Ag-bound scFv structure were calculated using PISA. The methyl a-D-mannopyranoside was procured from Sigma-Aldrich, and Coordinates and structure factors for the reported crystal structures have the dodecapeptide, DVFYPYPYASGS, was synthesized as described ear- been deposited in Research Collaboratory for Structural Bioinformatics lier (24). In brief, the dodecapeptide was synthesized by solid-phase Protein Data Bank (http://www.rcsb.org) under accession codes 4H0G (scFv), method on automated peptide synthesizer (431A; Applied Biosystems, 4H0I (scFv–sugar), and 4H0H (scFv–peptide). Foster City, CA) using 9-fluorenylmethyloxycarbonyl chemistry on a p- hydroxymethylphenoxymethyl polystyrene resin (Nova Biochem, San Diego, CA). Cleavage was performed using trifluoroacetic acid (Sigma-Aldrich),

Results Downloaded from and a Delta Pak C18 column (Waters, Milford, MA) was used to purify scFv 2D10: the Ig variable domain crude peptide preparation using a linear gradient of acetonitrile containing 0.1% trifluoroacetic acid. Identity of the purified peptide was established The variable fragment of the mAb 2D10 was constructed in the format by mass spectroscopy. of single-chain Ab (scFv 2D10). The scFv protein was expressed without any terminal tag-tail (26). The scFv 2D10, in the absence of scFv expression, purification, and crystallization Ag and in complex with either methyl a-D-mannopyranoside or the For Ab protein expression, recombinant plasmid pVEX-2D10 was used. The mimicking dodecapeptide, DVFYPYPYASGS, crystallized in space http://www.jimmunol.org/ scFv protein was prepared by in vitro refolding from its misfolded aggregates group P3 21 with one molecule per asymmetric unit. Structures of as described (26). In brief, recombinant plasmid pVEX-2D10–transformed 1 BL 21(DE3) cells were cultured at 37˚C, and protein expression was induced scFv in Ag-free state and in complex with the sugar or peptide were with 1 mM isopropyl b-D-thiogalactopyranoside. The scFv inclusion bodies determined by the molecular replacement method. The data and were solubilized in buffer containing 8 M urea and purified on a carbox- refinement statistics are presented in Table I, and the Ca repre- ymethyl-Sepharose (cation exchanger) column under denaturing conditions. sentations of the scFv models are shown in Fig. 1A. The final refined Purified scFv was subsequently subjected to refolding at 4˚C by gradual structures of scFv 2D10, in Ag-free and in Ag-bound forms, consist removal of urea, glutathione (oxidized), and L-arginine as described. The refolded protein was further concentrated at 15˚C to achieve concentrations of residues 1–124 of the H chain and 140–251 of the L chain. generally required for setting up crystallization trials. As shown in Fig. 1A and 1B, all three scFv structures exhibited

The scFv crystals in Ag-free and in complex with methyl a-D-man- standard Ig folds, one each for the H and the L chains. All six CDR by guest on September 28, 2021 nopyranoside were obtained under identical conditions at 4˚C in hanging loops and the two disulfide bridges of the scFv showed well-defined drops from mixtures containing a 1:1 ratio of scFv solution (12 mg/ml) and electron densities, whereas the linker residues could only be partly reservoir solution (100 mM MES [pH 6.5], 1.6 M MgSO4). Before setting up the crystallization of the complex, the scFv was incubated with sugar at three deciphered. In the case of the native scFv structure, six residues different molar ratios (1:20, 1:10, and 1:5) overnight at 4˚C. The complex of corresponding to the linker molecule could be modeled, whereas in scFv and sugar obtained at the 1:20 molar ratio produced diffraction quality the cases of the scFv–sugar complex and the scFv–peptide complex, crystals. In the case of the scFv–peptide complex, co-crystals were obtained at one and six residues, respectively, could be fitted in the electron 4˚C in hanging drops from mixtures containing a 1:1 ratio of scFv solution density. Alternatively, although a clearly discernible Ag density cor- (15 mg/ml) and reservoir solution (50 mM MES [pH 6.5], 1.6 M MgSO4). Prior to setting up the crystallization, scFv was preincubated with the peptide responding to the entire sugar molecule was observed, in the case at three different molar ratios (1:15, 1:10, and 1:5) overnight at 4˚C. The of the dodecapeptide, only 7 (FYPYPYA) of 12 residues could be scFv–peptide complexation achieved at a 1:15 molar ratio produced crystals interpreted and modeled (Fig. 1C), with an occupancy of 0.8. of diffraction quality. Appropriate size crystals of scFv, in Ag-free as well as in Ag-bound forms, were obtained over a period of 1 mo. Diffraction of scFv Structural features of scFv in Ag-free and Ag-bound states crystals (in complex with sugar and the peptide) after 1 mo (since the setting up of crystallization) produced complete data sets of 2.4 and 2.0 A˚ reso- The Ca backbones of all three structures of scFv 2D10 were super- lutions, respectively. However, the native scFv crystals, obtained along with imposed with root-mean-square deviations (rmsds) ranging from the scFv–sugar complex (under identical conditions), could be successfully 0.2to0.56A˚ with no striking difference in the overall structure of diffracted only after 1 y. The crystals of native scFv produced a complete data scFv among the three cases. The CDRs of the Ag-free and Ag- setata2.2A˚ resolution. bound scFv were also compared by superimposing all of the CDRs Data collection and structure determination of the H and L chains. The rmsd values for Ca atom positions of the , ˚ Complete diffraction data sets from cryoprotected (100% paraffin oil), flash- CDRs were found to be 0.5 A, and rmsd values for all atom frozen scFv 2D10 native and Ag-bound co-crystals were collected at a positions of the CDRs were ,1.0 A˚ (Fig. 2A). Superimposition of home source x-ray machine on a MAR345dtb image plate (Marresearch, the individual CDR loops among the Ag-free and Ag-bound struc- Norderstedt, Germany) installed on a rotating anode x-ray source (Rigaku, tures, as well as the superimposition between the sugar and the Tokyo, Japan) operating at 5 kW. The diffraction data sets were further pro- peptide-bound structures, exhibited similar results for both the Ca cessed using MOSFLM (27) and SCALA (28). The structure of the scFv–sugar complex was solved by molecular replacement using MolRep (29) by utiliz- positions and all atom positions of CDRs. However, the rmsd value ing whole-chain A (comprising VH–VL) from another scFv 1696 structure for the superimposition of all atom positions of the CDRL1 among (Brookhaven Protein Data Bank code 1JP5) as the search model. native scFv and the scFv–peptide complex was slightly higher at For the subsequent structure solution of the scFv in complex with pep- 1.2 A˚ . This increase in the observed rmsd value was contributed tide, chain A of the scFv 2D10–sugar complex was used, and for the Ab 163 structure in an Ag-free state, chain B of the scFv–peptide complex was mostly by the side chain of scFv residue Arg , which probably used as the search model. In each of these cases, the structure models were owing to being in a noninteracting state adopted different side chain refined using the Crystallography and NMR System (30). Coot (31) was orientations in native and Ag-complexed structures. Structural The Journal of Immunology 3

Table I. Data collection and refinement statistics

scFv scFv–Sugar scFv–Peptide Data collectiona Space group P3121 P3121 P3121 Cell dimensions a, b, c (A˚ ) 79.98, 79.98, 72.30 80.3, 80.3, 75.1 80.3, 80.3, 75.0 Resolution (A˚ ) 36.1–2.2 (2.32–2.20) 35.4–2.4 (2.53–2.40) 27.4–2.0 (2.11–2.0) b Rmerge 7.7 (59.7) 12.9 (66.6) 6.5 (42.7) I/sI 26.3 (4.6) 15.9 (3.2) 24.1 (5.5) Completeness (%) 100.0 (100.0) 100.0 (100.0) 100.0 (100.0) Redundancy 14.1 (14.0) 9.2 (9.2) 10.5 (10.1) Refinement Resolution (A˚ ) 35.0–2.3 34.0–2.4 25.0–2.0 No. reflections 12,216 11,290 17,376 c Rwork/Rfree 25.77/27.22 22.77/23.98 19.5/21.5 No. atoms Protein 1852 1836 1848 Ligand/ion 8 13/4 66 Water 180 133 204 B factors Protein 43.61 33.08 27.04 Downloaded from Ligand/ion 17.73 63.10/30.5 92.1 Water 48.43 38.18 38.9 rmsds Bond lengths (A˚ ) 0.013 0.010 0.028 Bond angles (˚) 2.06 1.725 1.865 aValues in parentheses are for the highest resolution shell. b Rmerge = S(I 2,I. )/SI, where I is the integrated intensity of each reflection. http://www.jimmunol.org/ c R value = S(|Fo | 2 |Fc |)/S|Fo|, where Fo and Fc are observed and calculated structure factor amplitudes. examination of the whole scFv molecule and its CDRs as a func- comparison with that observed in the native Ab structure. The tion of conformational change decisively established that the Ab rmsd values of superimposition of the Ca atoms and also the atom paratope is a rigid binding site, as it does not change conformation positions of this loop among Ag-free and the Ag-bound complexes upon Ag binding. were 1.4 and 2.6 A˚ , respectively. The conformation of the loop However, a loop (constituted by the VH residues 40–47) located was otherwise identical in both sugar and peptide-bound scFv just on the opposite face of the Ag combining site was observed complex structures with rmsd values of 0.2 A˚ between the Ca by guest on September 28, 2021 to adopt an altered conformation in the Ag-bound structures in positions and of 0.6 A˚ between all atom positions.

FIGURE 1. Structure of single-chain Ab 2D10 and its Ags. (A) scFv 2D10 in native form and in complex with Ags, namely methyl a-D-mannopyranoside and the dodecapeptide DVFYPYPYASGS. (B) Surface potential of scFv 2D10 in native and Ag-bound forms. (C) Ag structures in the scFv 2D10 complexes along with the corresponding 2Fo-Fc maps contoured at s cut- offof0.7. 4 PLASTICITY IN Ag–Ab INTERACTIONS Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. Stereo view of CDR loop conformations of the Ag-free and Ag-bound scFv molecules of scFv 2D10. (A) Superimposition of CDR regions of Ag-free scFv in complex with sugar and peptide scFv represented as smooth loops. The color codes are: magenta, scFv; yellow, scFv–sugar; blue, scFv–peptide. (B) CDR residues of scFv in complex with sugar; black dotted lines represent the hydrogen bonds between the Ab and Ag atoms. (C) CDR residues of scFv in complex with peptide; black dotted lines represent the hydrogen bonds between the Ab and Ag atoms.

Additionally, small interdomain movement at the VH–VL in- bonds, respectively. Among these hydrogen bonds, only five are terface of the scFv molecule was observed upon Ag binding. The common among the three scFv structures. Of the remaining hy- VH–VL interface of scFv, in Ag-free state and in complex with the drogen bonds, two are common at the VH–VL interface of scFv, sugar and the peptide, interacts through 10, 9, and 8 hydrogen which is in complex with the Ags. These observations suggest that The Journal of Immunology 5 a few hydrogen bonds break and a few new hydrogen bonds form extended toward the CDR H1 region (Figs. 1B, 2A). The core at the VH–VL interface upon Ag binding. However, these alter- region of the dodecapeptide forming a continuous epitope con- ations in the interaction do not change the paratope conformation. stituted 7 aa starting from the third up to the ninth residue (FYPYPYA) (Fig. 2C). Topological description of chemically dissimilar Ags of scFv The contacts of the sugar moiety with the Ab involved CDR H1, 2D10 L1, and L3 regions, primarily through five strong hydrogen bonds. Methyl a-D-mannopyranoside and the dodecapeptide, exhibiting Among all of the polar interactions, three were water-mediated functional mimicry as seen by the humoral immune system, were hydrogen bonds formed between various hydroxyl groups of the expected to have common structural features despite being chem- sugar moiety and the CDR H3 and L1 loops of the Ab. The O3 atom ically distinct. However, crystal structures of the Ag molecules of the hydroxyl group of mannose interacts with the hydroxyl group in complex with the scFv were found to have distinct surface of CDR L1 residue Tyr176 (Y176A), and the carbonyl oxygen of topologies. The scFv surfaces and interfaces were analyzed using CDR H3 residue Tyr102 (Y102A) interacted through a water mol- PISA (35). The peptide epitope had 3-fold larger surface area ecule by means of hydrogen bonds. Similarly, the O2 atom of (1065 A˚ 2) as compared with the sugar moiety with a surface area mannose interacted with the nitrogen (NE2) atom of CDR H3 of 327 A˚ 2 (Fig. 1C). Accordingly, the buried interfacial area be- residue Lys100 (K100A), and the O6 atom of sugar interacts with the tween Ag and the Ab was almost 3-fold more in the case of the carbonyl oxygen atom of CDR L3 residue Thr236 (T236A) through scFv–peptide complex (563 A˚ 2) than that which was observed in one water molecule each (Fig. 2B). Hence, it was evident that the case of the scFv–sugar complex (195 A˚ 2). Superimposition of sugar molecule was held tightly, mostly via water-mediated polar structures of scFv in complex with sugar to that with peptide interactions. Additionally, the scFv–sugar complex was also in- Downloaded from positioned the sugar moiety right over the Tyr8 of the peptide. volved in direct polar interactions between the sugar moiety and However, the phenol ring of the Tyr8 residue of peptide did not CDR L3 residues of the Ag combining site. The O4 and O6 atoms align completely over the sugar ring (Fig. 2A). The sugar moiety, of mannose formed hydrogen bonds with the hydroxyl and carbonyl which is a less flexible molecule compared with the dodecapeptide backbone oxygen atoms of the CDR L3 residues Ser235 (S235A) and which has a considerably large number of torsional angles, and Tyr240 (Y240A), respectively (Fig. 2B).

was found to fit more snugly than the peptide, as is evident from In contrast to the scFv–sugar interface interactions, peptide http://www.jimmunol.org/ the relatively low B factors (Table I). Beyond the obvious size exploited both polar as well as hydrophobic functional groups to differences between the carbohydrate and peptide molecules, contact Ab paratope. Besides CDR H3, L1, and L3 (similar to the shape disparity between the two Ags is also clearly evident. In sugar moiety), dodecapeptide epitope also interacts with a CDR H1 addition to size and shape differences, these two ligands belong to residue. The N-terminal half of the epitope was extensively in- two different classes of the biomolecules. That they are chemi- volved in forging hydrophobic contacts through p–p and lone cally distinct entities was also reflected in their interactions with pair–p electrons of the interacting residues. The phenyl rings of the the2D10Ableadingtomanifestation of functional mimicry peptide residues Tyr4 (Y4D) and Tyr6 (Y6D) stack over the phenyl without any structural similarity among the ligands. Alternatively, rings of the CDR H1 residue Tyr55 (Y55B) and CDRH3 residue

102 by guest on September 28, 2021 whereas methyl a-D-mannopyranoside interacted with Ab pri- Tyr (Y102B), respectively, forming strong p–p stacking inter- marily through polar interactions, dodecapeptide engaged both actions (Table II). Residue Phe3 (F3D) of the peptide was seen polar and hydrophobic interactions with the Ab to achieve func- interacting in a strategic manner, acting similar to a glue and holding tional mimicry (Table II). together the above two stacked phenyl ring pairs. The carbonyl oxygen of the Phe3 residue of peptide was observed to align itself Ag–Ab interface perpendicularly to the plane of the phenyl ring of the intramolecular In the scFv–carbohydrate complex, the entire sugar molecule was residue Tyr6 (Y6D), and its amide group and the phenyl ring were observed to make contact with the paratope (Fig. 2B). The sugar observed interacting with another intramolecular residue Tyr4 (Y4D) molecule was found to occupy the middle of the Ag combining phenol ring. As a result, three peptide residues, that is, Phe3,Tyr4, site of the Ab, precisely between the CDR H3 and L3 loops (Fig. and Tyr6, were streamlined and held together by two flanking ty- 2A). In the case of the scFv–peptide complex, the Tyr8 residue rosine residues of the paratope (Fig. 2C). (Y8D) toward the C terminus of the peptide positioned itself ex- The C-terminal half of the peptidyl epitope was found to interact actly over the sugar binding site, and the N terminus of the peptide with the paratope residues through polar interactions. Among the

Table II. Interactions of scFv 2D10 with sugar (AMG) and peptide (DVFYPYPYASGS) Ags

Peptide/Sugar Dist. (A˚ ) scFv/Water Peptide Polar interactions Tyr6 O 2.70 HOH61 O (Lys100: O, Dist. 2.5 A˚ ) Pro7 O 2.38 Ser105 OG Tyr8 N 3.57 Tyr240 OH Tyr8 OH 3.45 Tyr102 OH Tyr8 OH 2.87 Asn172 ND2 Nonpolar interactions Phe3 3.52 Tyr55 Tyr6 3.79 Tyr102 Sugar Polar interactions OH2 2.73 HOH131 O (Lys100: NE2, Dist. 2.32 A˚ ) OH3 2.76 HOH8 O (Tyr176: O, Dist. 2.63 A˚ ;Tyr102: O, Dist. 3.02 A˚ ) OH4 2.96 Ser235 OH OH6 2.80 Tyr240 OH OH6 3.43 HOH58 O (Thr236: OH, Dist. 3.10A˚ ) Residues in parentheses correspond to those residues involved in water-mediated hydrogen bonding. Dist., Distance. 6 PLASTICITY IN Ag–Ab INTERACTIONS six strong hydrogen bonds formed by this portion of the Ag, one was reactivities. Whereas the mAb 1H7 exhibited absolute specificity a water-mediated interaction. The carbonyl oxygen atoms of the for the immunogen at one end of the binding specificity spectrum, peptide residue Tyr6 (Y6D) and CDR H3 residue Lys100 (K100B) the mAb 2D10 exhibited reactivity to sugar and the 12-mer peptide interacted by forming hydrogen bonds through a water molecule. with equivalent affinities, reflecting immunological mimicry (24). Additional hydrogen bonds were formed between the carbonyl The 1H7 and 2D10 Abs were then analyzed for their conforma- oxygen of peptide Pro7 (P7D) and the side chain hydroxyl group tional attributes using thermodynamic studies and molecular dy- (OG) of the CDR H3 Ser105 (S105B); the amide nitrogen of peptide namics simulations. These analyses indicated a flexible paratope in Tyr8 (Y8D) and the CDR L3 Tyr240 (Y240B); and the hydroxyl the case of the multireactive 2D10 as opposed to 1H7, which was 8 group of the peptide Tyr (Y8D) with the hydroxyl group of CDR highly specific to methyl a-D-mannopyranoside. It was natural to H3 Tyr102 (Y102B) and the nitrogen atom of the side chain amino correlate the conformational flexibility of the 2D10 paratope with group (ND2) of CDR L1 Asn172 (N172B). Structural analysis of the its multireactivity (24, 25). However, in the absence of any direct interacting surfaces in the case of sugar and peptide Ags clearly structural evidence, the actual mechanism involved in the mani- revealed that both the moieties contact Ab through mutually inde- festation of the carbohydrate–peptide mimicry in the case of 2D10 pendent interactions. could not be unambiguously deciphered. The present structural Comparison of interactions involved in binding of sugar and studies were important precisely in this context. peptide with scFv demonstrated that neither the chemical nature nor The three crystal structures reported in this study illustrate the the equivalence in the interactions was the basis for the observed well-defined topology of the Ag combining site of the Ab 2D10. The mimicry. Interestingly, the mimicry observed in binding of the Ag combining site showed clear electron density and reasonable chemically and topologically unrelated molecules by 2D10 scFv temperature factors for all of the CDRs. The mannopyranoside-bound Downloaded from was simply achieved via plasticity of the Ag–Ab interactions without scFv paratope showed binding of the sugar in the region where li- any role for conformational changes of the CDR loops at the Ag gands bind in most of the carbohydrate binding Abs. In other known combining site. It was thus inferred, as depicted in Fig. 3, that the structures of Ab–carbohydrate complexes, the sugar binding region mutually independent set of interactions could result in binding of begins from the H3 loop and extends toward the H1 and H2 loops structurally as well as chemically different molecules through a (37–39). The region where mannopyranoside binds in scFv 2D10

combination of common as well as independent sets of residues in overlaps with this at one end. The dodecapeptide, which spanned http://www.jimmunol.org/ the Ag binding site. a larger area at the binding site in comparison with the sugar moiety, was adequately overlapping with the sugar binding site. Discussion Additionally, a portion of the bound peptide extended beyond the Molecular mimicry has been perceived as the breach of specificity overlapping region toward the H1 and H2 loops just as in ex- and has long been associated with autoimmune disorders and cancers tended oligosaccharide binding sites (40). The sugar molecule (12, 13). Even though the immune system encounters mimicry very utilizes primarily polar contacts involving merely six residues often, it does not always result in autoimmunity (36). Hence, the while binding to the Ab. The Tyr8 of the peptide binds at the mechanisms by which the immune system addresses the structural region where the sugar molecule sits and shares three important equivalences at the molecular level are of great relevance. While residues of the paratope involved in sugar binding. However, the by guest on September 28, 2021 addressing the mimicry between Tyr/Pro/Tyr-containing peptides number and nature of the bonds made between the Ab and and methyl a-D-mannopyranoside using crystallographic as well peptide are different. Peptide formed one water-mediated and as immunological methods (20–23), it was suggested that a motif four direct hydrogen bonds with the Ab. within the dodecapeptide could behave similar to a carbohydrate It is evident from the current crystallographic investigations that moiety at the structural level. Crystallographic analyses of the two mimicry has been achieved in the absence of topological equivalence molecules in complex with Con A showed them binding at two between the two chemically distinct moieties binding at the over- distinct sites, eluding any structural correlation between them (23). lapping surface using almost mutually independent interactions. Alternatively, the functional mimicry between the sugar and the Crystal structures of the scFv 2D10 in all three forms—without Ag, peptide was demonstrated from the cross-reactivity of the poly- in complex with sugar, and in complex with peptide—have revealed clonal sera raised against a-D-mannopyranoside and the dodeca- that the paratope of the Ab is structurally invariant, as represented peptide. Furthermore, each Ag triggered a cross-boosting secondary schematically in Fig. 3. Interestingly, observed structural invariance response in favor of the other (21). Therefore, the mystery of the while binding to two chemically distinct Ags is contrary to the mimicry between the sugar and the peptide remained unresolved, inferences determined on the basis of thermodynamic and modeling motivating explorations involving mAbs against them. studies (24, 25). Both the thermodynamic data and molecular dy- In an earlier study, the hybridoma clones generated against namic simulations provide clues for the possible conformational methyl a-D-mannopyranoside exhibited a wide range of ligand flexibility of 2D10, which was assumed to have been used toward binding different Ags. However, the present study elucidates 2D10 exhibiting multireactivity in a conformational state while interacting with two structurally different Ags. The interesting aspect of our study is that even if the potential for flexibility existed, it has not been used while recognizing two independent ligands, a sugar and a peptide, by 2D10. The two ligands are being recognized at the same level of affinity at the same combining site using different sets of interactions. In other words, the crystallographic data provide further insights in terms of elucidating polyreactivity through dif- ferent modes of binding without actually contradicting the potential FIGURE 3. Schematic representation of scFv molecule in Ag-free and of the Ab paratope for adopting multiple conformational states. It Ag-bound forms. The invariant Ag combining site of the 2D10 scFv does not violate any physicochemical principles of molecular rec- structure bound to structurally independent Ags using mutually indepen- ognition and yet provides a novel scenario of plasticity of inter- dent interactions. actions of a binding site. The Journal of Immunology 7

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