Govaerts C, Blanpain C, Deupi X, Ballet S, Ballesteros J A, Wodak S J, Vassart G, Pardo L, Parmentier M
Institut de Recherche Interdisciplinaire en Biologie Humaine et Nucléaire, Université Libre de Bruxelles, Campus Erasme, 808 route de Lennik, B-1070 Bruxelles, Belgium.
J Biol Chem. 2001 Apr 20;276(16):13217-25. doi: 10.1074/jbc.M011670200. Epub 2001 Jan 25.
CCR5 is a G-protein-coupled receptor activated by the chemokines RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein 1alpha and 1beta, and monocyte chemotactic protein 2 and is the main co-receptor for the macrophage-tropic human immunodeficiency virus strains. We have identified a sequence motif (TXP) in the second transmembrane helix of chemokine receptors and investigated its role by theoretical and experimental approaches. Molecular dynamics simulations of model alpha-helices in a nonpolar environment were used to show that a TXP motif strongly bends these helices, due to the coordinated action of the proline, which kinks the helix, and of the threonine, which further accentuates this structural deformation. Site-directed mutagenesis of the corresponding Pro and Thr residues in CCR5 allowed us to probe the consequences of these structural findings in the context of the whole receptor. The P84A mutation leads to a decreased binding affinity for chemokines and nearly abolishes the functional response of the receptor. In contrast, mutation of Thr-82(2.56) into Val, Ala, Cys, or Ser does not affect chemokine binding. However, the functional response was found to depend strongly on the nature of the substituted side chain. The rank order of impairment of receptor activation is P84A > T82V > T82A > T82C > T82S. This ranking of impairment parallels the bending of the alpha-helix observed in the molecular simulation study.
CCR5是一种G蛋白偶联受体，可被趋化因子RANTES（正常T细胞激活时表达和分泌的调节因子）、巨噬细胞炎性蛋白1α和1β以及单核细胞趋化蛋白2激活，是嗜巨噬细胞型人类免疫缺陷病毒株的主要共受体。我们在趋化因子受体的第二个跨膜螺旋中鉴定出一个序列基序（TXP），并通过理论和实验方法研究了其作用。在非极性环境中对模型α螺旋进行分子动力学模拟，结果表明由于脯氨酸使螺旋扭结以及苏氨酸进一步加剧这种结构变形的协同作用，TXP基序会强烈弯曲这些螺旋。对CCR5中相应的Pro和Thr残基进行定点诱变，使我们能够在整个受体的背景下探究这些结构发现的后果。P84A突变导致对趋化因子的结合亲和力降低，几乎消除了受体的功能反应。相比之下，将Thr-82（2.56）突变为Val、Ala、Cys或Ser并不影响趋化因子的结合。然而，发现功能反应强烈依赖于取代侧链的性质。受体激活受损的顺序为P84A > T82V > T82A > T82C > T82S。这种受损程度的排序与分子模拟研究中观察到的α螺旋弯曲情况相似。
