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PDBsum entry 2ywz

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Immune system PDB id
2ywz
Jmol
Contents
Protein chain
111 a.a. *
Waters ×119
* Residue conservation analysis
PDB id:
2ywz
Name: Immune system
Title: Structure of new antigen receptor variable domain from sharks
Structure: New antigen receptor variable domain. Chain: a. Engineered: yes
Source: Orectolobus maculatus. Spotted wobbegong. Organism_taxid: 168098. Expressed in: escherichia coli. Expression_system_taxid: 562.
Resolution:
2.21Å     R-factor:   0.189     R-free:   0.254
Authors: V.A.Streltsov
Key ref:
D.P.Simmons et al. (2008). Shark IgNAR antibody mimotopes target a murine immunoglobulin through extended CDR3 loop structures. Proteins, 71, 119-130. PubMed id: 17932913 DOI: 10.1002/prot.21663
Date:
23-Apr-07     Release date:   30-Oct-07    
PROCHECK
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 Headers
 References

Protein chain
Pfam   ArchSchema ?
A9CBG5  (A9CBG5_9CHON) -  New antigen receptor variable domain (Fragment)
Seq:
Struc:
111 a.a.
111 a.a.
Key:    PfamA domain  Secondary structure  CATH domain

 

 
DOI no: 10.1002/prot.21663 Proteins 71:119-130 (2008)
PubMed id: 17932913  
 
 
Shark IgNAR antibody mimotopes target a murine immunoglobulin through extended CDR3 loop structures.
D.P.Simmons, V.A.Streltsov, O.Dolezal, P.J.Hudson, A.M.Coley, M.Foley, D.F.Proll, S.D.Nuttall.
 
  ABSTRACT  
 
Mimotopes mimic the three-dimensional topology of an antigen epitope, and are frequently recognized by antibodies with affinities comparable to those obtained for the original antibody-antigen interaction. Peptides and anti-idiotypic antibodies are two classes of protein mimotopes that mimic the topology (but not necessarily the sequence) of the parental antigen. In this study, we combine these two classes by selecting mimotopes based on single domain IgNAR antibodies, which display exceptionally long CDR3 loop regions (analogous to a constrained peptide library) presented in the context of an immunoglobulin framework with adjacent and supporting CDR1 loops. By screening an in vitro phage-display library of IgNAR variable domains (V(NAR)s) against the target antigen monoclonal antibody MAb5G8, we obtained four potential mimotopes. MAb5G8 targets a linear tripeptide epitope (AYP) in the flexible signal sequence of the Plasmodium falciparum Apical Membrane Antigen-1 (AMA1), and this or similar motifs were detected in the CDR loops of all four V(NAR)s. The V(NAR)s, 1-A-2, -7, -11, and -14, were demonstrated to bind specifically to this paratope by competition studies with an artificial peptide and all showed enhanced affinities (3-46 nM) compared to the parental antigen (175 nM). Crystallographic studies of recombinant proteins 1-A-7 and 1-A-11 showed that the SYP motifs on these V(NAR)s presented at the tip of the exposed CDR3 loops, ideally positioned within bulge-like structures to make contact with the MAb5G8 antibody. These loops, in particular in 1-A-11, were further stabilized by inter- and intra- loop disulphide bridges, hydrogen bonds, electrostatic interactions, and aromatic residue packing. We rationalize the higher affinity of the V(NAR)s compared to the parental antigen by suggesting that adjacent CDR1 and framework residues contribute to binding affinity, through interactions with other CDR regions on the antibody, though of course definitive support of this hypothesis will rely on co-crystallographic studies. Alternatively, the selection of mimotopes from a large (<4 x 10(8)) constrained library may have allowed selection of variants with even more favorable epitope topologies than present in the original antigenic structure, illustrating the power of in vivo selection of mimotopes from phage-displayed molecular libraries.
 
  Selected figure(s)  
 
Figure 1.
Figure 1. Isolation of MAb5G8-specific V[NAR] domains. (A) Pools of polyclonal IgNAR-bacteriophages displaying V[NAR] domains were assayed for binding to MAb5G8 (selection antigen) or 2% MPBS (nonspecific control) pre-pan and following one to four rounds of library panning. Bound bacteriophages were detected using an HRP conjugated anti-fd phage antibody, and results represent the average of triplicate wells. The positive control represents bacteriophages displaying the MAb5G8-specific E2 peptide [^NEDENTLQHAYPID^C]. (B) Protein alignment of deduced amino acid sequences for V[NAR]s 1-A-2, 1-A-7, 1-A-11, and 1-A-14. CDR1 and CDR3 regions are highlighted and identical residues (dark shading) and conservative replacements (light shading; I/V/L/M, D/E, K/R, A/G, T/S, Q/N, F/Y) indicated. (C) Expanded comparison of V[NAR] 1-A-2, 1-A-7, 1-A-11, and 1-A-14 CDR regions. Potential peptide motifs for interaction with MAb5G8 are underlined, and 1-A-11 cysteine residues positioned to form an inter-loop disulphide linkage are highlighted in bold.
Figure 4.
Figure 4. Comparative analysis of V[NAR] loop topologies. (A) Superimposed VMD[58] ribbon representations of V[NAR] domain crystallographic structures. Expanded CDR3 protein backbone traces are shown in ribbon representation for Type 2 V[NAR]s 1-A-7 (green; this study), 1-A-11 (red; this study), 12A-9 (cyan; PDB 2COQ), 12Y-2 (yellow; PDB 1VES), and the Type 1 V[NAR] HEL-5A7 (blue; PDB 1SQ2). (B) Licorice representations of the 1-A-7 CDR3 loop highlighting the potential mimotope at residues ^95SYP. (C) Ribbon representation of the 1-A-7 CDR3 (green) and CDR1 (yellow) loop regions in 180° rotation from (B). Loop stabilizing interactions are shown as dotted lines. (D) Licorice representations of the 1-A-11 CDR3 loop highlighting the potential mimotope at residues ^93SYP. (E) Ribbon representation of the 1-A-11 CDR3 (green) and CDR1 (yellow) loop regions in 180° rotation from (C). Loop stabilizing interactions are shown, including the inter-loop disulphide bond between Cys^29 and Cys^91.
 
  The above figures are reprinted by permission from John Wiley & Sons, Inc.: Proteins (2008, 71, 119-130) copyright 2008.  
  Figures were selected by the author.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
19196718 A.Monegal, D.Ami, C.Martinelli, H.Huang, M.Aliprandi, P.Capasso, C.Francavilla, G.Ossolengo, and A.de Marco (2009).
Immunological applications of single-domain llama recombinant antibodies isolated from a naïve library.
  Protein Eng Des Sel, 22, 273-280.  
19529959 J.Wesolowski, V.Alzogaray, J.Reyelt, M.Unger, K.Juarez, M.Urrutia, A.Cauerhff, W.Danquah, B.Rissiek, F.Scheuplein, N.Schwarz, S.Adriouch, O.Boyer, M.Seman, A.Licea, D.V.Serreze, F.A.Goldbaum, F.Haag, and F.Koch-Nolte (2009).
Single domain antibodies: promising experimental and therapeutic tools in infection and immunity.
  Med Microbiol Immunol, 198, 157-174.  
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