Bondue Antoine, Jao Shu-chuan, Blanpain Cédric, Parmentier Marc, LiWang Patricia J
IRIBHN, Université Libre de Bruxelles, Campus Erasme, 808 route de Lennik, B-1070 Bruxelles, Belgium.
Biochemistry. 2002 Nov 19;41(46):13548-55. doi: 10.1021/bi026087d.
MIP-1beta is a CC-chemokine that plays a role in inflammation and host defense mechanisms by interacting with its specific receptor CCR5. CCR5 is a major coreceptor for macrophage-tropic human immunodeficiency virus (HIV), and as a consequence, MIP-1beta can inhibit HIV entry. It is therefore of interest to understand how MIP-1beta and other CCR5 ligands bind to their receptor, as such understanding could lead to the rational design of more efficient HIV entry blockers. We have previously demonstrated the importance of Phe13, and of basic residues of the 40's loop, in mediating high-affinity binding of MIP-1beta to CCR5. We have now investigated further the relative contribution of other MIP-1beta residues in the interaction of the chemokine with CCR5, by studying the functional consequences of point mutations within the N-loop and the 3(10) turn of MIP-1beta, affecting the charge, size, and H-bonding properties of the side chains. Our data suggest that, in addition to Phe13, three amino acids of the N-loop and 3(10) turn (Arg18, Lys19, and Arg22) interact with CCR5 through their positive charge. We also found that Pro21 contributes to the CCR5 binding properties of MIP-1beta. Moreover, NMR spectroscopy has revealed that the presence of Tyr at position 15 is necessary for the proper folding of the chemokine. Our results therefore demonstrate that the binding determinants of MIP-1beta consist of residues arranged on one surface of the protein, including most of the basic residues in MIP-1beta, as well as two key hydrophobic groups. The good correlation observed between the potency of the mutants in a functional assay and their binding affinity strongly argues that basic residues Arg18, Lys19, and Arg22 of MIP-1beta are essential for its CCR5 binding properties, without a primary effect on CCR5 activation.
巨噬细胞炎性蛋白-1β（MIP-1β）是一种CC趋化因子，通过与其特异性受体CCR5相互作用，在炎症和宿主防御机制中发挥作用。CCR5是嗜巨噬细胞型人类免疫缺陷病毒（HIV）的主要共受体，因此，MIP-1β可抑制HIV进入。所以，了解MIP-1β和其他CCR5配体如何与其受体结合很有意义，因为这样的了解可能会带来更高效的HIV进入阻滞剂的合理设计。我们之前已经证明了苯丙氨酸13以及40环的碱性残基在介导MIP-1β与CCR5的高亲和力结合中的重要性。我们现在通过研究MIP-1β的N环和3（10）转角内点突变的功能后果，进一步研究了其他MIP-1β残基在趋化因子与CCR5相互作用中的相对贡献，这些点突变影响侧链的电荷、大小和氢键性质。我们的数据表明，除了苯丙氨酸13外，N环和3（10）转角的三个氨基酸（精氨酸18、赖氨酸19和精氨酸22）通过其正电荷与CCR5相互作用。我们还发现脯氨酸21有助于MIP-1β的CCR5结合特性。此外，核磁共振光谱显示，第15位酪氨酸的存在对于趋化因子的正确折叠是必要的。因此，我们的结果表明，MIP-1β的结合决定因素由排列在蛋白质一个表面上的残基组成，包括MIP-1β中的大多数碱性残基以及两个关键的疏水基团。在功能测定中观察到的突变体效力与其结合亲和力之间的良好相关性有力地表明，MIP-1β的碱性残基精氨酸18、赖氨酸19和精氨酸22对其CCR5结合特性至关重要，而对CCR5激活没有主要影响。
