Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128, USA.
Proteins. 2010 Feb 1;78(2):295-308. doi: 10.1002/prot.22542.
The N-terminal region of the chemokine RANTES is critical for its function. A synthesized N-terminally modified analog of RANTES, P2-RANTES, was discovered using a phage display selection against living CCR5-expressing cells, and has been reported to inhibit HIV-1 env-mediated cell-cell fusion at subnanomolar levels (Hartley et al. J Virol 2003;77:6637-6644). In the present study we produced this protein using E. coli overexpression and extensively studied its structure and function. The x-ray crystal structure of P2-RANTES was solved and refined at 1.7 A resolution. This protein was found to be predominantly a monomer in solution by analytical ultracentrifugation, but a tetramer in the crystal. In studies of glycosaminoglycan binding, P2-RANTES was found to be significantly less able to bind heparin than wild type RANTES. We also tested this protein for receptor internalization where it was shown to be functional, in cell-cell fusion assays where recombinant P2-RANTES was a potent fusion inhibitor (IC(50) = 2.4 +/- 0.8 nM), and in single round infection assays where P2-RANTES inhibited at subnanomolar levels. Further, in a modified fusion assay designed to test specificity of inhibition, P2-RANTES was also highly effective, with a 65-fold improvement over the fusion inhibitor C37, which is closely related to the clinically approved inhibitor T-20. These studies provide detailed structural and functional information for this novel N-terminally modified chemokine mutant. This information will be very useful in the development of more potent anti-HIV agents. PDB Accession Number: 2vxw.
趋化因子 RANTES 的 N 端区域对其功能至关重要。使用针对表达活 CCR5 细胞的噬菌体展示筛选，发现了趋化因子 RANTES 的 N 端修饰类似物 P2-RANTES，并已报道其能以亚纳摩尔水平抑制 HIV-1 env 介导的细胞-细胞融合（Hartley 等人，J Virol 2003；77:6637-6644）。在本研究中，我们使用大肠杆菌过表达生产了这种蛋白质，并对其结构和功能进行了深入研究。通过 X 射线晶体学解析了 P2-RANTES 的晶体结构，分辨率为 1.7Å。通过分析超速离心实验发现该蛋白在溶液中主要以单体形式存在，但在晶体中以四聚体形式存在。在糖胺聚糖结合研究中，发现 P2-RANTES 与野生型 RANTES 相比，结合肝素的能力显著降低。我们还测试了该蛋白的受体内化功能，结果表明其在细胞-细胞融合实验中具有功能，在重组 P2-RANTES 作为有效的融合抑制剂（IC50=2.4±0.8 nM）的实验中，以及在单轮感染实验中，P2-RANTES 以亚纳摩尔水平抑制病毒感染。此外，在设计用于测试抑制特异性的改良融合实验中，P2-RANTES 也非常有效，与密切相关的临床批准抑制剂 T-20 相比，抑制效果提高了 65 倍。这些研究为这种新型 N 端修饰趋化因子突变体提供了详细的结构和功能信息。这些信息将对开发更有效的抗 HIV 药物非常有用。PDB 访问编号：2vxw。
