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 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 28, 2021 Subscription Information about subscribing to The Journal of Immunology 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 © 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,