Structure of AMA1 from Plasmodium falciparum reveals a clustering of polymorphisms that surround a conserved hydrophobic pocket

Abstract

Apical membrane antigen 1 (AMA1) is a leading malaria vaccine candidate that possesses polymorphisms that may pose a problem for a vaccine based on this antigen. Knowledge of the distribution of the polymorphic sites on the surface of AMA1 is necessary to obtain a detailed understanding of their significance for vaccine development. For this reason we have sought to determine the three-dimensional structure of AMA1 using x-ray crystallography. The central two-thirds of AMA1 is relatively conserved among Plasmodium species as well as more distantly related apicomplexan parasites, and contains two clusters of disulfide-bonded cysteines termed domains I and II. The crystal structure of this fragment of AMA1 reported here reveals that domains I؉II consists of two intimately associated PAN domains. PAN domain I contains many long loops that extend from the domain core and form a scaffold for numerous polymorphic residues. This extreme adaptation of a PAN domain reveals how malaria parasites have introduced significant flexibility and variation into AMA1 to evade protective human antibody responses. The polymorphisms on the AMA1 surface are exclusively located on one side of the molecule, presumably because this region of AMA1 is most accessible to antibodies reacting with the parasite surface. Moreover, the most highly polymorphic residues surround a conserved hydrophobic trough that is ringed by domain I and domain II loops. Precedents set by viral receptor proteins would suggest that this is likely to be the AMA1 receptor binding pocket.

Far from the Madding Crowd: the Molecular Basis for Immunological Escape ofPlasmodium falciparum

“…The numbers for the amino acids in the CSP-1 and TRAP TSR domain sequences are from the encoding system reported by Rodriguez et al, 2008. ! 69 cHABPs of Plasmodium falciparum mediated (after prodomain removal) by AMA-1 domains I and II where cHABPs 4313 ( 134 DAEVAGTQYRLPSGKCPVFG 153 ) (DI) and 4325 ( 374 MIKSAFLPTGAFKADRYKSH 393 ) (DII) form a trough or channel stabilised by H-bonds where an as-yet-unknown receptor binds ( Figure 2C, blue and dark yellow) (Bai et al, 2005).…”

“…The crystal structure of Pf Rh5 bound to growthinhibitory monoclonal antibody fragments 9AD4 (PDB code 4U0R) is also shown (Wright et al, 2014). (C1) Pf AMA-1 recombinant fragment (PDB accession number 1Z40) (Bai et al, 2005) containing domains I (residues 108-308) and II (residues 309-436) and the location of RBCbinding cHABPs 4313 (blue) and 4325 (dark yellow), displaying the trough where a still undetermined RBC receptor binds. A view of the nonpolymorphic and polymorphic face of this protein is also shown, as well as the 3D structure of Pf AMA-1 complexed with 1F9 mAb (PDB accession number 2Q8A) (Coley et al, 2007).…”

“…T h e s e polymorphic residues surround a hydrophobic pocket suggested as being critical for AMA-1 function. Domain I has the major polymorphic sites, grouped into 3 clusters (C1, C2 and C3); regarding C1-L (loop containing residues 196, 197, 199, 200, 201, 204, 206 and 207), 1F9 mAb binds in loop Id and most human antibodies react against it (Bai et al, 2005;Dutta et al, 2007;Takala et al, 2009;Ouattara et al, 2013;Harris et al, 2014). However, once the epitope has been recognised it inhibits in vitro invasion in a strain-specific manner, as in the case of 1F9 mAb inhibiting in vitro growth of 3D7 and D10 parasite strains, but not HB3 or W2mef where a single aa substitution at the most highly polymorphic site (residue 197) in AMA-1 abolishes 1F9 mAb binding (Coley et al, 2006), thereby limiting the effectiveness of these antigens as vaccine components.…”

Population structure of the genes encoding the polymorphic Plasmodium falciparum apical membrane antigen 1: Implications for vaccine design

“…Despite its overall polymorphism, PfAMA1 remains a leading vaccine candidate for several reasons. First, the structure (two Pan domains) and the location of disulfide bridges are highly conserved in all species of Plasmodium (4,5). Second, targeted disruption of the PfAMA1 gene has not been successful, indicating that the protein is critical for blood-stage survival of the parasite (6).…”

The crystal structure of P. knowlesi DBPα DBL domain and its implications for immune evasion

“…In such a mechanism of immune escape polymorphic residues near the binding site elude binding of inhibitory antibody thus protecting the critical functional site on the ligand domain. Previously, this type of mechanism similar to influenza hemagglutinin was proposed for the P. vivax DBP [16][17][18] and recent structural data suggests that polymorphisms of another Plasmodium microneme ligand, apical membrane antigen 1 (AMA1), also serve to evade immune antibody inhibition [19] . …”