The TXP motif in the second transmembrane helix of CCR5 - A structural determinant of chemokine-induced activation

CCR5 is a G-protein-coupled receptor activated by the chemokines RANTES (re gulated on activation normal T cell expressed and secreted), macrophage inf lammatory protein Icu and Ip, and monocyte chemotactic protein 2 and is the main co-receptor for the macrophage-tropic human immunodeficiency virus st rains. We have identified a sequence motif (TXP) in the second transmembran e helix of chemokine receptors and investigated its role by theoretical and experimental approaches. Molecular dynamics simulations of model cy-helice s in a nonpolar environment were used to show that a TXP motif strongly ben ds 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 re sidues in CCR5 allowed us to probe the consequences of these structural fin dings in the context of the whole receptor. The P84A mutation leads to a de creased 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 sid e 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.