The Ig new antigen receptors (IgNARs) are single-domain antibodies found in the serum of sharks. Here, we report 2.2-and 2.8-Å structures of the type 2 IgNAR variable domains 12Y-1 and 12Y-2. Structural features include, first, an Ig superfamily topology transitional between cell adhesion molecules, antibodies, and T cell receptors; and, second, a vestigial complementarity-determining region 2 at the ''bottom'' of the molecule, apparently discontinuous from the antigen-binding paratope and similar to that observed in cell adhesion molecules. Thus, we suggest that IgNARs originated as cell-surface adhesion molecules coopted to the immune repertoire and represent an evolutionary lineage independent of variable heavy chain͞variable light chain type antibodies. Additionally, both 12Y-1 and 12Y-2 form unique crystallographic dimers, predominantly mediated by main-chain framework interactions, which represent a possible model for primordial cell-based interactions. Unusually, the 12Y-2 complementarity-determining region 3 also adopts an extended ␤-hairpin structure, suggesting a distinct selective advantage in accessing cryptic antigenic epitopes.

The new antigen receptor (IgNAR) is an antibody unique to sharks and consists of a disulphide-bonded dimer of two protein chains, each containing a single variable and five constant domains. The individual variable (V(NAR)) domains bind antigen independently, and are candidates for the smallest antibody-based immune recognition units. We have previously produced a library of V(NAR) domains with extensive variability in the CDR1 and CDR3 loops displayed on the surface of bacteriophage. Now, to test the efficacy of this library, and further explore the dynamics of V(NAR) antigen binding we have performed selection experiments against an infectious disease target, the malarial Apical Membrane Antigen-1 (AMA1) from Plasmodium falciparum. Two related V(NAR) clones were selected, characterized by long (16- and 18-residue) CDR3 loops. These recombinant V(NAR)s could be harvested at yields approaching 5mg/L of monomeric protein from the E. coli periplasm, and bound AMA1 with nanomolar affinities (K(D)= approximately 2 x 10(-7) M). One clone, designated 12Y-2, was affinity-matured by error prone PCR, resulting in several variants with mutations mapping to the CDR1 and CDR3 loops. The best of these variants showed approximately 10-fold enhanced affinity over 12Y-2 and was Plasmodium falciparum strain-specific. Importantly, we demonstrated that this monovalent V(NAR) co-localized with rabbit anti-AMA1 antisera on the surface of malarial parasites and thus may have utility in diagnostic applications.

The ecdysone receptor is a hormone-dependent transcription factor that plays a central role in regulating the expression of vast networks of genes during development and reproduction in the phylum Arthropoda. The functional receptor is a heterodimer of the two nuclear receptor proteins ecdysone receptor (EcR) and ultraspiracle protein. The receptor is the target of the environmentally friendly bisacylhydrazine insecticides, which are effective against Lepidoptera but not against Hemiptera or several other insect orders. Here we present evidence indicating that much of the selectivity of the bisacylhydrazine insecticides can be studied at the level of their binding to purified ecdysone receptor ligand-binding domain (LBD) heterodimers. We report the crystal structure of the ecdysone receptor LBD heterodimer of the hemipteran Bemisia tabaci (Bt, sweet potato whitefly) in complex with the ecdysone analogue ponasterone A. Although comparison with the corresponding known LBD structure from the lepidopteran Heliothis virescens (Hv) ecdysone receptor revealed the overall mode of ponasterone A binding to be very similar in the two cases, we observed that the BtEcR ecdysteroid-binding pocket is structured differently to that of HvEcR in those parts that are not in contact with ponasterone A. We suggest that these differences in the ligand-binding pocket may provide a molecular basis for the taxonomic order selectivity of bisacylhydrazine insecticides. The nuclear receptor (NR)1 family of proteins plays a crucial role in the regulation of transcription, and its members include the receptors for steroid hormones, vitamins, thyroid hormones, and bile acids (1). The human genome contains about 48 members of this family, and these have been studied extensively as therapeutic targets (2). The Arthropoda display a more limited suite of NRs (3) about 21 of which occur in Drosophila melanogaster. Among these is the receptor for the major arthropod steroid hormone, 20-hydroxyecdysone, which is involved in the regulation of insect molting, metamorphosis, and reproduction (4 -9). The receptor is absent from mammals and is thus potentially useful as a safe insecticide target. Indeed members of the bisacylhydrazine family exert their insecticidal activity by binding to the ecdysone receptor and exhibit remarkable taxonomic order selectivity (10, 11). These compounds act selectively on the Lepidoptera and certain Coleoptera (10) but are ineffective against insects of the hemipteran order and therefore cannot be used to control certain insect pests. A study (12) of two hemipteran insect predators (Geocoris punctipes and Orius insidiosus) showed that these beneficial (predatory) hemipterans are relatively insensitive to the bisacylhydrazine tebufenozide, whereas lepidopteran insect pests are susceptible. An improved understanding of variation in the structure of the ligand-binding pockets of ecdysone receptors and the basis of the specificity of these compounds at the atomic level of detail of their interaction with the receptor may aid ...

The new antigen receptor (IgNAR) antibodies from sharks are disulphide bonded dimers of two protein chains, each containing one variable and five constant domains. Three types of IgNAR variable domains have been discovered, with Type 3 appearing early in shark development and being overtaken by the antigen-driven affinity-matured Type 1 and 2 response. Here, we have determined the first structure of a naturally occurring Type 2 IgNAR variable domain, and identified the disulphide bond that links and stabilizes the CDR1 and CDR3 loops. This disulphide bridge locks the CDR3 loop in an "upright" conformation in contrast to other shark antibody structures, where a more lateral configuration is observed. Further, we sought to model the Type 3 isotype based on the crystallographic structure reported here. This modeling indicates (1) that internal Type 3-specific residues combine to pack into a compact immunoglobulin core that supports the CDR loop regions, and (2) that despite apparent low-sequence variability, there is sufficient plasticity in the CDR3 loop to form a conformationally diverse antigen-binding surface.Keywords: immunoglobulin new antigen receptor; single variable domain; antibody; paratope; shark; cell adhesion molecule Supplemental material: see www.proteinscience.orgImmunoglobulin new antigen receptors (IgNARs) are a unique antibody isotype found in the serum of sharks (Greenberg et al. 1995;Nuttall et al. 2001). They are bivalent, but target antigen through a single immunoglobulin variable domain (,13 kDa) displaying two complementarity determining region (CDR) loops (Roux et al. 1998;Nuttall et al. 2003). In contrast, conventional antibodies have a variable heavy (V H ) + variable light (V L ) domain format (,26 kDa) and bind antigen through up to six CDRs (Chothia et al. 1989;Padlan 1994). To compensate for their reduced size, IgNARs encode unusually long and structurally complex CDR3s, which display a high degree of variability (Greenberg et al. 1995;Nuttall et al. 2004).To date, three IgNAR isotypes have been identified, which vary in the number and configuration of their framework cysteine residues, and time of appearance in shark development (Rumfelt et al. 2002). Type 3 IgNARs, the last discovered, display limited diversity in both the size Abbreviations: IgNAR, new antigen receptor antibody from sharks; V NAR , single variable domain of the IgNAR antibody; CDR, complementarity determining region; kDa, kilodalton.Article published online ahead of print. Article and publication date are at