Structure of an IgNAR-AMA1 Complex: Targeting a Conserved Hydrophobic Cleft Broadens Malarial Strain Recognition

Abstract

Apical membrane antigen 1 (AMA1) is essential for invasion of erythrocytes and hepatocytes by Plasmodium parasites and is a leading malarial vaccine candidate. Although conventional antibodies to AMA1 can prevent such invasion, extensive polymorphisms within surface-exposed loops may limit the ability of these AMA1-induced antibodies to protect against all parasite genotypes. Using an AMA1-specific IgNAR single-variable-domain antibody, we performed targeted mutagenesis and selection against AMA1 from three P. falciparum strains. We present cocrystal structures of two antibody-AMA1 complexes which reveal extended IgNAR CDR3 loops penetrating deep into a hydrophobic cleft on the antigen surface and contacting residues conserved across parasite species. Comparison of a series of affinity-enhancing mutations allowed dissection of their relative contributions to binding kinetics and correlation with inhibition of erythrocyte invasion. These findings provide insights into mechanisms of single-domain antibody binding, and may enable design of reagents targeting otherwise cryptic epitopes in pathogen antigens.

Antigen recognition by single-domain antibodies: structural latitudes and constraints

“…Henderson et al . 69 suggested that recognition of a conserved hydrophobic cleft on Plasmodium AMA1 by a V NAR (12Y-2 and its affinity-matured variants) reflected a novel binding mode; although the epitope of a murine conventional antibody (1F9) substantially overlapped that of V NAR 12Y-2, 1F9 binding depended to a greater degree on polymorphic loop residues surrounding the hydrophobic trough. Likewise, Ditlev et al .…”

“…Additional examples of binding to recessed epitopes on proteins (clefts, cavities, crevices or grooves) can be found for sdAbs against lactococcal siphophage, 97 Plasmodium falciparum MTIP, 98 epidermal growth factor receptor, 99 and respiratory syncytial virus fusion protein, 100 although in these cases it is less clear that these sites are inaccessible to conventional antibodies. While it is possible that V NAR s may share similar cleft-binding proclivities, such claims are based on very limited published data (three structures 7,12,69 ). Moreover, it should be noted that there are many examples (not covered in this review) of partial or complete overlap between the epitopes of sdAbs and conventional antibodies, and thus the degree to which sdAbs bind cryptic epitopes vs .…”

Crystal Structure of the Amyloid-β p3 Fragment Provides a Model for Oligomer Formation in Alzheimer's Disease

“…For A␤-IgNAR-G1, note replacement of A␤ Ala42 with glycine, which modeling suggested was required to allow sufficient rotational freedom for realignment of the loop structure. A␤-IgNAR gene constructs were produced by splice-overlap PCR as previously described (Henderson et al, 2007) using IgNAR 12Y-2 DNA template and A␤ oligonucleotide primers (supplemental Table S1, available at www.jneurosci.org as supplemental material). DNA cassettes were cloned into Escherichia coli periplasmic expression vector pGC as described (Henderson et al, 2007).…”

Therapeutically targeting glypican-3 via a conformation-specific single-domain antibody in hepatocellular carcinoma

“…However, none of these antibodies has shown the ability to inhibit cell proliferation or induce apoptosis, possibly because of the difficulty of having a conventional antibody targeting the potentially cryptic functional epitope of GPC3. Because of their small size, domain antibodies are able to target cryptic epitopes on antigens (e.g., in the clefts of enzymes and receptors) (28)(29)(30).…”