Menendez Alfredo, Calarese Daniel A, Stanfield Robyn L, Chow Keith C, Scanlan Chris N, Kunert Renate, Katinger Herman, Burton Dennis R, Wilson Ian A, Scott Jamie K
Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada.
FASEB J. 2008 May;22(5):1380-92. doi: 10.1096/fj.07-8983com. Epub 2008 Jan 15.
MAb 2G12 neutralizes HIV-1 by binding with high affinity to a cluster of high-mannose oligosaccharides on the envelope glycoprotein, gp120. Screening of phage-displayed peptide libraries with 2G12 identified peptides that bind specifically, with K(d)s ranging from 0.4 to 200 microM. The crystal structure of a 21-mer peptide ligand in complex with 2G12 Fab was determined at 2.8 A resolution. Comparison of this structure with previous structures of 2G12-carbohydrate complexes revealed striking differences in the mechanism of 2G12 binding to peptide vs. carbohydrate. The peptide occupies a site different from, but adjacent to, the primary carbohydrate-binding site on 2G12, and makes only slightly fewer contacts to the Fab than Man(9)GlcNAc(2) (51 vs. 56, respectively). However, only two antibody contacts with the peptide are hydrogen bonds in contrast to six with Man(9)GlcNAc(2), and only three of the antibody residues that interact with Man(9)GlcNAc(2) also contact the peptide. Thus, this mechanism of peptide binding to 2G12 does not support structural mimicry of the native carbohydrate epitope on gp120, since it neither replicates the oligosaccharide footprint on the antibody nor most of the contact residues. Moreover, 2G12.1 peptide is not an immunogenic mimic of the 2G12 epitope, since antisera produced against it did not bind gp120.
单克隆抗体2G12通过与包膜糖蛋白gp120上的一组高甘露糖寡糖高亲和力结合来中和HIV-1。用2G12筛选噬菌体展示肽库,鉴定出特异性结合的肽,其解离常数(K(d))范围为0.4至200微摩尔。以2.8埃的分辨率确定了与2G12 Fab复合的21聚体肽配体的晶体结构。将该结构与2G12-碳水化合物复合物的先前结构进行比较,发现2G12与肽和碳水化合物结合机制存在显著差异。该肽占据的位点与2G12上的主要碳水化合物结合位点不同但相邻,与Fab的接触比Man(9)GlcNAc(2)仅略少(分别为51次和56次)。然而,与肽的抗体接触中只有两个是氢键,而与Man(9)GlcNAc(2)有六个,并且与Man(9)GlcNAc(2)相互作用的抗体残基中只有三个也与肽接触。因此,这种肽与2G12的结合机制不支持gp120上天然碳水化合物表位的结构模拟,因为它既不复制抗体上的寡糖足迹,也不复制大多数接触残基。此外,2G12.1肽不是2G12表位的免疫原性模拟物,因为针对它产生的抗血清不与gp120结合。