Kanwal J. Kaur

Topological Analysis of the Functional Mimicry between a Peptide and a Carbohydrate Moiety

The shared surface topology of two chemically dissimilar but functionally equivalent molecular structures has been analyzed. A carbohydrate moiety (α-D-mannopyranoside) and a peptide molecule (DVFYPYPYASGS) bind to concanavalin A at a common binding site. The cross-reactivity of the polyclonal antibodies (pAbs) was used for understanding the topological relationship between these two independent ligands. The anti-α-D-mannopyranoside pAbs recognized various peptide ligands of concanavalin A, and the anti-DVFYPYPYASGS pAbs recognized the carbohydrate ligands, providing direct evidence of molecular mimicry. On the basis of differential binding of various rationally designed peptide analogs to the anti-α-D-mannopyranoside pAbs, it was possible to identify different peptide residues critical for the mimicry. The comparison of circular dichroism profiles of the designed analogs suggests that the carbohydrate mimicking conformation of the peptide ligand incorporates a polyproline type II structural fold. The concanavalin A binding activity of these analogs was found to have a direct correlation with the topological relationship between peptide and carbohydrate ligands.

Structure-Function Analysis of Tritrypticin, an Antibacterial Peptide of Innate Immune Origin

The structural requirements for the antibacterial activity of a pseudosymmetric 13-residue peptide, tritrypticin, were analyzed by combining pattern recognition in protein structures, the structure-activity knowledge-base, and circular dichroism. The structure-activity analysis, based on various deletion analogs, led to the identification of two minimal functional peptides, which by themselves exhibit adequate antibacterial activity againstEscherichia coli and Salmonella typhimurium. The common features between these two peptides are that they both share an aromatic-proline-aromatic (ArProAr) sequence motif, and their sequences are retro with respect to one another. The pattern searches in protein structure data base using the ArProAr motif led to the identification of two distinct conformational clusters, namely polyproline type II and β-turn, which correspond to the observed solution structures of the two minimal functional analogs. The role of different residues in structure and function of tritrypticin was delineated by analyzing antibacterial activity and circular dichroism spectra of various designed analogs. Three main results arise from this study. First, the ArProAr sequence motif in proteins has definitive conformational features associated with it. Second, the two minimal bioactive domains of tritrypticin have entirely different structures while having equivalent activities. Third, tritrypticin has a β-turn conformation in solution, but the functionally relevant conformation of this gene-encoded peptide antibiotic may be an extended polyproline type II.

Plasticity in structure and interactions is critical for the action of indolicidin, an antibacterial peptide of innate immune origin

The comparative analysis of two cationic antibacterial peptides of the cathelicidin family—indolicidin and tritrypticin—enabled addressing the structural features critical for the mechanism of indolicidin activity. Functional behavior of retro‐indolicidin was found to be identical to that of native indolicidin. It is apparent that the gross conformational propensities associated with retro‐peptides resemble those of the native sequences, suggesting that native and retro‐peptides can have similar structures. Both the native and the retro‐indolicidin show identical affinities while binding to endotoxin, the initial event associated with the antibacterial activity of cationic peptide antibiotics. The indolicidin–endotoxin binding was modeled by docking the indolicidin molecule in the endotoxin structure. The conformational flexibility associated with the indolicidin residues, as well as that of the fatty acid chains of endotoxin combined with the relatively strong structural interactions, such as ionic and hydrophobic, provide the basis for the endotoxin–peptide recognition. Thus, the key feature of the recognition between the cationic antibacterial peptides and endotoxin is the plasticity of molecular interactions, which may have been designed for the purpose of maintaining activity against a broad range of organisms, a hallmark of primitive host defense.

Functional Equality in the Absence of Structural Similarity AN ADDED DIMENSION TO MOLECULAR MIMICRY

The crystal structure ofmeso-tetrasulfonatophenylporphyrin complexed with concanavalin A (ConA) was determined at 1.9 Å resolution. Comparison of this structure with that of ConA bound to methyl α-d-mannopyranoside provided direct structural evidence of molecular mimicry in the context of ligand receptor binding. The sulfonatophenyl group of meso-tetrasulfonatophenylporphyrin occupies the same binding site on ConA as that of methyl α-d-mannopyranoside, a natural ligand. A pair of stacked porphyrin molecules stabilizes the crystal structure by end-to-end cross-linking with ConA resulting in a network similar to that observed upon agglutination of cells by lectins. The porphyrin binds to ConA predominantly through hydrogen bonds and water-mediated interactions. The sandwiched water molecules in the complex play a cementing role, facilitating favorable binding of porphyrin. Seven of the eight hydrogen bonds observed between methyl α-d-mannopyranoside and ConA are mimicked by the sulfonatophenyl group of porphyrin after incorporating two water molecules. Thus, the similarity in chemical interactions was manifested in terms of functional mimicry despite the obvious structural dissimilarity between the sugar and the porphyrin.

Mimicry of native peptide antigens by the corresponding retro-inverso analogs is dependent on their intrinsic structure and interaction propensities.

Retro-inverso (ri) analogs of model T cell and B cell epitopes were predictively designed as mimics and then assayed for activity to understand the basis of functional ri-antigenic peptide mimicry. ri versions of two MHC class I binding peptide epitopes, one from a vesicular stomatitis virus glycoprotein (VSVp) and another from OVA (OVAp), exhibit structural as well as functional mimicry of their native counterparts. The two ri peptides exhibit conformational plasticity and they bind to MHC class I (H-2Kb) similar to their native counterparts both in silico and in vivo. In fact, ri-OVAp is also presented to an OVAp-specific T cell line in a mode similar to native OVAp. In contrast, the ri version of an immunodominant B cell peptide epitope from a hepatitis B virus protein, PS1, exhibits no structural or functional correlation with its native counterpart. PS1 and its ri analog do not exhibit similar conformational propensities. PS1 is less flexible relative to its ri version. These observed structure-function relationships of the ri-peptide epitopes are consistent with the differences in recognition properties between peptide-MHC vs peptide-Ab binding where, while the recognition of the epitope by MHC is pattern based, the exquisitely specific recognition of Ag by Ab arises from the high complementarity between the Ag and the binding site of the Ab. It is evident that the correlation of conformational and interaction propensities of native l-peptides and their ri counterparts depends both on their inherent structural properties and on their mode of recognition.

Plasticity within the Antigen-Combining Site May Manifest as Molecular Mimicry in the Humoral Immune Response

Structural and physiological facets of carbohydrate-peptide mimicry were addressed by analyzing the Ab response to α-d-mannopyranoside. mAbs against α-d-mannopyranoside were generated and screened with the carbohydrate-mimicking 12 mer (DVFYPYPYASGS) peptide. Three mAbs, 2D10, 1H11, and 1H7, which were subjected to detailed analysis, exhibit diverse V gene usage, indicating their independent germline origins. Although the mAb 1H7 was specific in binding only to the immunizing Ag, the Abs 2D10 and 1H11 recognize the 12 mer peptide as well as the immunogen, α-d-mannopyranoside. The Abs that recognize mimicry appear to bind to a common epitope on the peptide and do not share the mode of peptide binding with Con A. Binding kinetics and thermodynamics of Ag recognition suggest that the Ab that does not recognize peptide-carbohydrate mimicry probably has a predesigned mannopyranoside-complementing site. In contrast, the mimicry-recognizing Abs adopt the Ag-combining site only on exposure to the sugar, exploiting the conformational flexibility in the CDRs. Although the mAb 1H7 showed unique specificity toward mannopyranoside, the mimicry-recognizing Abs 2D10 and 1H11 exhibited degenerate specificities with regard to other sugar moieties. It is proposed that the degeneracy of specificity arising from the plasticity at the Ag-combining site in a subset of the Ab clones may be responsible for exhibiting molecular mimicry in the context of Ab response.

Plasticity in Protein-Peptide Recognition: Crystal Structures of Two Different Peptides Bound to Concanavalin A

The structures of concanavalin A (ConA) in complex with two carbohydrate-mimicking peptides, 10-mer (MYWYPYASGS) and 15-mer (RVWYPYGSYLTASGS) have been determined at 2.75 A resolution. In both crystal structures four independent peptide molecules bind to each of the crystallographically independent subunits of ConA tetramer. The peptides exhibit small but significant variability in conformations and interactions while binding to ConA. The crystal structure of another similar peptide, 12-mer (DVFYPYPYASGS), in complex with ConA has been determined (Jain, D., K. J. Kaur, B. Sundaravadivel, and D. M. Salunke. 2000. Structural and functional consequences of peptide-carbohydrate mimicry. J. Biol. Chem. 275:16098-16102). Comparison of the three complexes shows that the peptides bind to ConA at a common binding site, using different contacting residues and interactions depending on their sequence and the local environment at the binding site. The binding is also optimized by corresponding plasticity of the peptide binding site on ConA. The diversity in conformation and interactions observed here are in agreement with the structural leeway concerning plasticity of specific molecular recognition in biological processes. The adaptability of peptide-ConA interactions may also be correlated with the carbohydrate-mimicking property of these peptides.

Porphyrin binding to jacalin is facilitated by the inherent plasticity of the carbohydrate-binding site: novel mode of lectin-ligand interaction.

The crystal structure of the complex of meso-tetrasulfonatophenylporphyrin (H(2)TPPS) with jack fruit (Artocarpus integriflora) agglutinin (jacalin) has been determined at 1.8 A resolution. A porphyrin pair is sandwiched between two symmetry-related jacalin monomers in the crystal, leading to a cross-linking network of protein molecules. Apart from the stacking interactions, H(2)TPPS also forms hydrogen bonds, some involving water bridges, with jacalin at the carbohydrate-binding site. The residues that are involved in rendering galactopyranoside specificity to jacalin undergo conformational adjustments in order to accommodate the H(2)TPPS molecule. The water molecules at the carbohydrate-binding site of jacalin cement the jacalin-porphyrin interactions, optimizing their complementarity. Interactions of porphyrin with jacalin are relatively weak compared with those observed between galactopyranoside and jacalin, perhaps because the former largely involves water-mediated hydrogen bonds. While H(2)TPPS binds to jacalin at the carbohydrate-binding site as in the case of ConA, its mode of interaction with jacalin is very different. H(2)TPPS does not enter the carbohydrate-binding cavity of jacalin. Instead, it sits over the binding site. While the porphyrin binding is mediated by replicating the hydrogen-bonding network of mannopyranoside through the sulfonate atoms in the case of ConA, the plasticity associated with the carbohydrate-binding site accommodates the pluripotent porphyrin molecule in the case of jacalin through an entirely different set of interactions.

Identification and characterization of EhCaBP2: a second member of the calcium-binding protein family of the protozoan parasite entamoeba histolytica

Entamoeba histolytica, an early branching eukaryote, is the etiologic agent of amebiasis. Calcium plays a pivotal role in the pathogenesis of amebiasis by modulating the cytopathic properties of the parasite. However, the mechanistic role of Ca2+ and calcium-binding proteins in the pathogenesis of E. histolytica remains poorly understood. We had previously characterized a novel calcium-binding protein (EhCaBP1) from E. histolytica. Here, we report the identification and partial characterization of an isoform of this protein, EhCaBP2. Both EhCaBPs have four canonical EF-hand Ca2+ binding domains. The two isoforms are encoded by genes of the same size (402 bp). Comparison between the two genes showed an overall identity of 79% at the nucleotide sequence level. This identity dropped to 40% in the 75-nucleotide central linker region between the second and third Ca2+ binding domains. Both of these genes are single copy, as revealed by Southern hybridization. Analysis of the available E. histolytica genome sequence data suggested that the two genes are non-allelic. Homology-based structural modeling showed that the major differences between the two EhCaBPs lie in the central linker region, normally involved in binding target molecules. A number of studies indicated that EhCaBP1 and EhCaBP2 are functionally different. They bind different sets of E. histolytica proteins in a Ca2+-dependent manner. Activation of endogenous kinase was also found to be unique for the two proteins and the Ca2+ concentration required for their optimal functionality was also different. In addition, a 12-mer peptide was identified from a random peptide library that could differentially bind the two proteins. Our data suggest that EhCaBP2 is a new member of a class of E. histolytica calcium-binding proteins involved in a novel calcium signal transduction pathway.

Immunological implications of structural mimicry between a dodecapeptide and a carbohydrate moiety

The immunogenicity and antibody cross-reactivity of two chemically different but structurally equivalent molecular mimics were analyzed by presenting them to the immune system in different modes. The observed differences in IgM and IgG responses in terms of cross-reactivity with the mimicking antigen could reflect the differential proliferative abilities of the corresponding B-cells. Modification of the T-cell help, either by using a promiscuous T-cell epitope or by prepriming, led to the shift in the antibody response towards the mimicking epitope. Also, the anti-sugar antibodies could be boosted using a carbohydrate mimicking peptide on cross-immunization. Thus, the carbohydrate-peptide mimicry appears to be a topological quasi-equivalence reflected differently in terms of antibody response during maturation.