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.

SUMMARYMild proteolysis by trypsin of particles of six potyviruses (bean yellow mosaic virus, clover yellow vein virus, Johnson grass mosaic virus, passion-fruit woodiness virus, potato virus Y and watermelon mosaic virus II) revealed that the N-and C-terminal regions of their coat protein are exposed on the particles' surfaces. The enzyme treatment removed the N-terminal region (30 to 67 amino acids long, depending on the virus) and 18 to 20 amino acids from the C terminus of the coat proteins, leaving a fully assembled virus particle composed of coat protein cores consisting of 216 or 218 amino acid residues. These core particles were indistinguishable from untreated native particles in an electron microscope and were still infectious. The core particles lacked the virus-specific surface epitopes that are recognized by the bulk of the polyclonal antibodies raised against the whole virus particles. Epitopes thought to be groupspecific were located in the trypsin-resistant core protein region. The implications of these findings are discussed in relation to the similar surface location of the N-and Cterminal regions of the coat protein of other rod-shaped plant viruses and the observed common structural features displayed by isometric plant and animal viruses.

The human insulin receptor is a homodimer consisting of two monomers linked by disulfide bonds. Each monomer comprises an ␣-chain that is entirely extracellular and a ␤-chain that spans the cell membrane. The ␣-chain has a total of 37 cysteine residues, most of which form intrachain disulfide bonds, whereas the ␤-chain contains 10 cysteine residues, four of which are in the extracellular region. There are two classes of disulfide bonds in the insulin receptor, those that can be reduced under mild reducing conditions to give ␣-␤ monomers (class I) and those that require stronger reducing conditions (class II). The number of class I disulfides is small and includes the ␣-␣ dimer bond Cys 524 . In this report we describe the use of cyanogen bromide and protease digestion of the exon 11 plus form of the receptor ectodomain to identify disulfide linkages between the ␤-chain residues Cys 798 and Cys 807 and between the ␣-chain Cys 647 and the ␤-chain Cys 872 . The latter bond is the sole ␣-␤ link in the molecule and implies a side-byside alignment of the two fibronectin III domains of the receptor. Also presented is evidence for additional ␣-␣ dimer bond(s) involving at least one of the cysteine residues of the triplet at positions 682, 683, and 685. Evidence is also presented to show that Cys 884 exists as a buried thiol in the soluble ectodomain. The insulin receptor (IR)1 is a homodimer, (␣␤) 2 , held together by disulfide bonds. Each ␣␤ monomer comprises an ␣-chain that is entirely extracellular and a ␤-chain that spans the cell membrane via a single transmembrane link to an intracellular segment that includes the protein-tyrosine kinase domain and the domains involved in binding signal transduction proteins (1). The ␣␤ monomer of the IR is coded for by 22 exons (2), is synthesized with a 27-residue signal sequence, and is glycosylated, S-S linked, and proteolytically processed in that order during transport to the cell surface (3, 4). The extracellular region of the mature receptor (the ectodomain) contains the ␣-chain (1-735) and 194 residues of the ␤-chain (the ␤Ј-chain, residues 736 -929). Two isoforms of the IR, which arise by alternative splicing of the mRNA, are expressed in a tissue-specific fashion; they differ by the presence (plus exon 11) or the absence (minus exon 11) of a 12-residue segment close to the C terminus of the ␣-chain (5).The ectodomain of the IR has been shown (6, 7) to contain two homologous domains (L1 and L2) separated by a single cysteine-rich region (residues 159 -310). The L2 domain is joined by a connecting domain (residues 471-593) to the Cterminal portion (from residue 594) that is comprised of two fibronectin (Fn) III repeats, the first of which contains an insert domain that includes both the ␣-␤ cleavage site and the alternately spliced exon 11 (8, 9). A schematic diagram showing the relative arrangement of the domains is shown in Fig. 7.The ␣-chain has a total of 37 cysteine residues, whereas the ␤-chain has 4 extracellular and 6 intracellular cysteine residues. Homologous re...