Keizer David W, Miles Luke A, Li Felomena, Nair Margie, Anders Robin F, Coley Andrew M, Foley Michael, Norton Raymond S
The Walter & Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia.
Biochemistry. 2003 Aug 26;42(33):9915-23. doi: 10.1021/bi034376b.
Apical membrane antigen 1 (AMA1) of the human malaria parasite Plasmodium falciparum is synthesized by schizont stage parasites and has been implicated in merozoite invasion of host erythrocytes. Phage-display techniques have recently been used to identify two 15-residue peptides, F1 and F2, which bind specifically to P. falciparum AMA1 and inhibit parasite invasion of erythrocytes [Li, F., et al. (2002) J. Biol. Chem. 277, 50303-50310]. We have synthesized F1, F2, and three peptides with high levels of sequence identity, determined their relative binding affinities for P. falciparum AMA1 with a competition ELISA, and investigated their solution structures by NMR spectroscopy. The strongest binding peptide, F1, contains a beta-turn that includes residues identified via an alanine scan as being critical for binding to AMA1 and inhibition of merozoite invasion of erythrocytes. The three F1 analogues include a 10-residue analogue of F1 truncated at the C-terminus (tF1), a partially scrambled 15-mer (sF1), and a disulfide-constrained 14-mer (F1tbp) which is related to F1 but has a sequence identical to that of a disulfide-constrained loop in the first epidermal growth factor module of the latent transforming growth factor-beta binding protein. tF1 and F1tbp bound competitively with F1 to AMA1, and all three contain a type I beta-turn encompassing key residues involved in F1 binding. In contrast, sF1 lacked this structural motif, and did not compete for binding to AMA1 with F1; rather, sF1 contained a type III beta-turn involving a different part of the sequence. Although F2 was able to bind to AMA1, it was unstructured in solution, consistent with its weak invasion inhibitory effects. Thus, the secondary structure elements observed for these peptides in solution correlate well with their potency in binding to AMA1 and inhibiting merozoite invasion. The structures provide a valuable starting point for the development of peptidomimetics as antimalarial antagonists directed at AMA1.
人类疟原虫恶性疟原虫的顶端膜抗原1（AMA1）由裂殖体阶段的寄生虫合成，并与裂殖子侵入宿主红细胞有关。噬菌体展示技术最近被用于鉴定两种15个残基的肽，F1和F2，它们特异性结合恶性疟原虫AMA1并抑制寄生虫对红细胞的侵入[Li, F., 等人（2002年）《生物化学杂志》277, 50303 - 50310]。我们合成了F1、F2以及三种具有高度序列同一性的肽，通过竞争ELISA测定它们对恶性疟原虫AMA1的相对结合亲和力，并通过核磁共振光谱研究它们的溶液结构。结合力最强的肽F1含有一个β-转角，其中包括通过丙氨酸扫描确定的对于结合AMA1和抑制裂殖子侵入红细胞至关重要的残基。三种F1类似物包括在C末端截短的10个残基的F1类似物（tF1）、部分打乱的15聚体（sF1）以及与F1相关但序列与潜伏转化生长因子-β结合蛋白的第一个表皮生长因子模块中的二硫键约束环相同的二硫键约束的14聚体（F1tbp）。tF1和F1tbp与F1竞争结合AMA1，并且这三种都含有一个I型β-转角，包含参与F1结合的关键残基。相比之下，sF1缺乏这种结构基序，并且不与F1竞争结合AMA1；相反，sF1含有一个涉及序列不同部分的III型β-转角。尽管F2能够结合AMA1，但它在溶液中无结构，这与其较弱的侵入抑制作用一致。因此，在溶液中观察到的这些肽的二级结构元件与其结合AMA1和抑制裂殖子侵入的效力密切相关。这些结构为开发作为针对AMA1的抗疟拮抗剂的肽模拟物提供了有价值的起点。
