Paratope plasticity in diverse modes facilitates molecular mimicry in antibody response

The immune response against methyl-α -D-mannopyranoside mimicking 12-mer peptide (DVFYPYPYASGS) was analyzed at the molecular level towards understanding the equivalence of these otherwise disparate Ags. The Ab 7C4 recognized the immunizing peptide and its mimicking carbohydrate Ag with comparable affinities. Thermodynamic analyses of the binding interactions of both molecules suggested that the mAb 7C4 paratope lacks substantial conformational flexibility, an obvious possibility for facilitating binding to chemically dissimilar Ags. Favorable changes in entropy during binding indicated the importance of hydrophobic interactions in recognition of the mimicking carbohydrate Ag. Indeed, the topology of the Ag-combining site was dominated by a cluster of aromatic residues, contributed primarily by the specificity defining CDR H3. Epitope-mapping analysis demonstrated the critical role of three aromatic residues of the 12-mer in binding to the Ab. Our studies delineate a mechanism by which mimicry is manifested in the absence of either structural similarity of the epitopes or conformational flexibility in the paratope. An alternate mode of recognition of dissimilar yet mimicking Ags by the anti-peptide Ab involves plasticity associated with aromatic/hydrophobic and van der Waals interactions. Thus, antigenic mimicry may be a consequence of paratope-specific modulations rather than being dependent only on the properties of the epitope. Such modulations may have evolved toward minimizing the consequences of antigenic variation by invading pathogens

Putative Bioactive Motif of Tritrpticin Revealed by an Antibody with Biological Receptor-Like Properties

<div><p>Antimicrobial peptides represent one of the most promising future strategies for combating infections and microbial drug resistance. Tritrpticin is a 13mer tryptophan-rich cationic antimicrobial peptide with a broad spectrum of activity whose application in antimicrobial therapy has been hampered by ambiguity about its biological target and consequently the molecular interactions necessary for its antimicrobial activity. The present study provides clues about the mechanism of action of tritripticin by using a unique monoclonal antibody (mAb) as a ‘physiological’ structural scaffold. A pool of mAbs were generated against tritrpticin and based on its high affinity and ability to bind tritrpticin analogs, mAb 6C6D7 was selected and characterized further. In a screening of phage displayed random peptides, this antibody was able to identify a novel antimicrobial peptide with low sequence homology to tritrpticin, suggesting that the mAb possessed the physico-chemical characteristics mimicking the natural receptor. Subsequently, thermodynamics and molecular modeling identified a core group of hydrophobic residues in tritrpticin arranged in a distorted’s’ shaped conformation as critical for antibody binding. Comparison of the mAb induced conformation with the micelle bound structure of tritrpticin reveals how a common motif may be able to interact with multiple classes of biomolecules thus extending the target range of this innate immune peptide. Based on the concurrence between thermodynamic and structural data our results reveal a template that can be used to design novel antimicrobial pharmacophores while simultaneously demonstrating at a more fundamental level the potential of mAbs to act as receptor surrogates.</p> </div

Anions Mediate Ligand Binding in Adineta vaga Glutamate Receptor Ion Channels

SummaryAvGluR1, a glutamate receptor ion channel from the primitive eukaryote Adineta vaga, is activated by alanine, cysteine, methionine, and phenylalanine, which produce lectin-sensitive desensitizing responses like those to glutamate, aspartate, and serine. AvGluR1 LBD crystal structures reveal an unusual scheme for binding dissimilar ligands that may be utilized by distantly related odorant/chemosensory receptors. Arginine residues in domain 2 coordinate the γ-carboxyl group of glutamate, whereas in the alanine, methionine, and serine complexes a chloride ion acts as a surrogate ligand, replacing the γ-carboxyl group. Removal of Cl− lowers affinity for these ligands but not for glutamate or aspartate nor for phenylalanine, which occludes the anion binding site and binds with low affinity. AvGluR1 LBD crystal structures and sedimentation analysis also provide insights into the evolutionary link between prokaryotic and eukaryotic iGluRs and reveal features unique to both classes, emphasizing the need for additional structure-based studies on iGluR-ligand interactions

Binding analysis of 6C6D7.

<p>A. Direct binding ELISA demonstrating cross-reactivity of mAb 6C6D7 with N-terminal deletion analogs of tritrpticin. B. SPR sensorgram of binding of mAb (62.5-1000 nM) to K-tritrpticin on a CM-4 chip.</p

SPR based thermodynamics of antigen – antibody interaction.

<p>A. Changes in Gibbs free energy of binding (∆G) at different temperatures (15°C - 35°C). B. Arrhenius plot for association. C. Arrhenius plot for dissociation. D. Individual contributions of enthalpy (∆H) and entropy (T∆S) to equilibrium free energy of binding (∆G), at temperatures ranging from 15°C to 35°C. Bars corresponding to ∆H are not visible in the figure because of their negligible contribution.</p

Sequencing of 6C6D7 light and heavy chain variable regions.

<p>Amino acid sequence of 6C6D7 light and heavy chain variable regions are shown with CDRs marked. The CDRs were determined from the gene sequence using IMGT database and IgG BLAST.</p

Novel antibiotic peptides picked by anti-tritrpticin mAb 6C6D7.

<p>A. Alignment of tritrpticin with novel sequence panned from random peptide library by mAb 6C6D7. B. Comparison of antibiotic activities of tritrpticin and phage displayed peptide against <i>E. coli</i> BL21λD3 in a radial diffusion assay. An unrelated peptide EHGTPPRVMSSM was used as negative control and as expected did not led to the formation of clearance zone (not shown in the figure). 50 nmol of peptides were used in the assay.</p

<i>In- silico</i> binding analysis of tritrpticin to mAb 6C6D7.

<p>A. RMSDs of Cα atoms of the CDRs and tritrpticin (black) and that of whole 6C6D7 Fv and tritrpticin (red) during the 8 ns MD simulation. The averaged structure from last 6 ns run was energy minimized and used for further analysis. B. Surface view of peptide – mAb interaction, shaded according to electrostatic potential (blue to red indicating positive to negative, respectively) showing complementarity of shape and charge. Also can be seen the three tryptophans buried in the hydrophobic cavity shielded from solvent. C. Stereoscopic representation of binding pocket containing bound peptide. Interacting residues for peptide are shown as ribbon and for Fv as sticks. The polar interactions obtained by PISA are shown as dotted lines. D. Stereo view of the binding pocket showing preponderance of aromatic residues both in paratope (ribbon) and epitope (sticks).</p