Saphire Erica Ollmann, Montero Marinieve, Menendez Alfredo, van Houten Nienke E, Irving Melita B, Pantophlet Ralph, Zwick Michael B, Parren Paul W H I, Burton Dennis R, Scott Jamie K, Wilson Ian A
Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
J Mol Biol. 2007 Jun 8;369(3):696-709. doi: 10.1016/j.jmb.2007.01.060. Epub 2007 Jan 27.
The human antibody b12 recognizes a discontinuous epitope on gp120 and is one of the rare monoclonal antibodies that neutralize a broad range of primary human immunodeficiency virus type 1 (HIV-1) isolates. We previously reported the isolation of B2.1, a dimeric peptide that binds with high specificity to b12 and competes with gp120 for b12 antibody binding. Here, we show that the affinity of B2.1 was improved 60-fold over its synthetic-peptide counterpart by fusing it to the N terminus of a soluble protein. This affinity, which is within an order of magnitude of that of gp120, probably more closely reflects the affinity of the phage-borne peptide. The crystal structure of a complex between Fab of b12 and B2.1 was determined at 1.8 A resolution. The structural data allowed the differentiation of residues that form critical contacts with b12 from those required for maintenance of the antigenic structure of the peptide, and revealed that three contiguous residues mediate B2.1's critical contacts with b12. This single region of critical contact between the B2.1 peptide and the b12 paratope is unlikely to mimic the discontinuous key binding residues involved in the full b12 epitope for gp120, as previously identified by alanine scanning substitutions on the gp120 surface. These structural observations are supported by experiments that demonstrate that B2.1 is an ineffective immunogenic mimic of the b12 epitope on gp120. Indeed, an extensive series of immunizations with B2.1 in various forms failed to produce gp120 cross-reactive sera. The functional and structural data presented here, however, suggest that the mechanism by which b12 recognizes the two antigens is very different. Here, we present the first crystal structure of peptide bound to an antibody that was originally raised against a discontinuous protein epitope. Our results highlight the challenge of producing immunogens that mimic discontinuous protein epitopes, and the necessity of combining complementary experimental approaches in analyzing the antigenic and immunogenic properties of putative molecular mimics.
人源抗体b12识别gp120上的一个不连续表位，是中和多种原发性人类免疫缺陷病毒1型（HIV-1）分离株的罕见单克隆抗体之一。我们之前报道了B2.1的分离，它是一种二聚体肽，能与b12高特异性结合，并与gp120竞争b12抗体结合。在此，我们表明，通过将B2.1融合到一种可溶性蛋白的N端，其亲和力比其合成肽对应物提高了60倍。这种亲和力与gp120的亲和力在一个数量级内，可能更紧密地反映了噬菌体携带肽的亲和力。b12的Fab与B2.1之间复合物的晶体结构在1.8埃分辨率下得以确定。结构数据使得能够区分与b12形成关键接触的残基和维持肽抗原结构所需的残基，并揭示三个连续残基介导了B2.1与b12的关键接触。B2.1肽与b12互补位之间的这个关键接触单一区域不太可能模拟gp120完整b12表位中涉及的不连续关键结合残基，正如之前通过对gp120表面进行丙氨酸扫描替换所确定的那样。这些结构观察结果得到了实验的支持，这些实验表明B2.1是gp120上b12表位的无效免疫原性模拟物。事实上，用各种形式的B2.1进行的一系列广泛免疫未能产生gp120交叉反应血清。然而，此处呈现的功能和结构数据表明，b12识别这两种抗原的机制非常不同。在此，我们展示了与最初针对不连续蛋白质表位产生的抗体结合的肽的首个晶体结构。我们的结果突出了产生模拟不连续蛋白质表位的免疫原的挑战，以及在分析假定分子模拟物的抗原性和免疫原性特性时结合互补实验方法的必要性。
