Duma Luminita, Häussinger Daniel, Rogowski Marco, Lusso Paolo, Grzesiek Stephan
Division of Structural Biology, Biozentrum, University of Basel, Klingelbergstr. 70, Basel, Switzerland.
J Mol Biol. 2007 Jan 26;365(4):1063-75. doi: 10.1016/j.jmb.2006.10.040. Epub 2006 Oct 17.
The chemokine RANTES (regulated upon activation, normal T-cell expressed and secreted) is a natural ligand of CCR5, one of the major HIV-1 coreceptors. It is secreted as part of the immune response to human immunodeficiency virus 1 (HIV-1) and inhibits infection by CCR5-dependent (R5) HIV-1 isolates. We have investigated the interaction of RANTES with several peptides derived from the extracellular domains of CCR5 by heteronuclear NMR spectroscopy in aqueous solution. We show that a peptide comprising the first 25 amino acid residues of the CCR5 N-terminal domain and sulfated at the Y10 and Y14 side-chains binds with micromolar affinity exclusively to the monomeric form of RANTES. In contrast to the tight binding of the sulfated peptide, the affinity of the same peptide in non-sulfated form was reduced by more than two orders of magnitude. Peptides derived from the CCR5 extracellular loops ECL1, ECL2 and ECL3 showed only very moderate and mostly non-specific binding. Chemical shift mapping of the interaction of the sulfated N-terminal peptide reveals a contiguous binding surface on RANTES, which comprises amino acid residues of the first beta-strand, the N-loop, the fourth beta-strand and the turns around residues 30 and 40. This binding surface largely overlaps with the dimer interface and is strongly positively charged, providing a rationale for the exclusive binding of the monomer to the peptide and the requirement of the negative sulfate groups at the Y10 and Y14 side-chains. The binding surface also largely overlaps with the segments that were identified previously as crucial for HIV blockade by peptide scanning and mutagenesis studies. These data offer new insights into the structure-function relation of the RANTES-CCR5 interaction and may be helpful for the design of novel HIV-1 inhibitors.
趋化因子RANTES（活化时受调控、正常T细胞表达和分泌）是主要的HIV-1共受体之一CCR5的天然配体。它作为对人类免疫缺陷病毒1（HIV-1）免疫反应的一部分被分泌，并抑制CCR5依赖性（R5）HIV-1分离株的感染。我们通过水溶液中的异核核磁共振光谱研究了RANTES与源自CCR5细胞外结构域的几种肽的相互作用。我们发现，一种包含CCR5 N端结构域前25个氨基酸残基且在Y10和Y14侧链处硫酸化的肽，以微摩尔亲和力仅与RANTES的单体形式结合。与硫酸化肽的紧密结合相反，相同肽的非硫酸化形式的亲和力降低了两个多数量级。源自CCR5细胞外环ECL1、ECL2和ECL3的肽仅表现出非常适度且大多是非特异性的结合。硫酸化N端肽相互作用的化学位移图谱揭示了RANTES上一个连续的结合表面，该表面包含第一条β链、N环、第四条β链以及残基30和40周围的转角处的氨基酸残基。这个结合表面在很大程度上与二聚体界面重叠，且带强正电荷，这为单体与肽的特异性结合以及Y10和Y14侧链上负硫酸基团的必要性提供了理论依据。该结合表面也在很大程度上与先前通过肽扫描和诱变研究确定的对HIV阻断至关重要的片段重叠。这些数据为RANTES-CCR5相互作用的结构-功能关系提供了新的见解，可能有助于新型HIV-1抑制剂的设计。
