The TXP Motif in the Second Transmembrane Helix of CCR5

Abstract

CCR5 is a G-protein-coupled receptor activated by the chemokines RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein 1␣ and 1␤, 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 ␣-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 ␣-helix observed in the molecular simulation study.

Constitutive Activation of CCR5 and CCR2 Induced by Conformational Changes in the Conserved TXP Motif in Transmembrane Helix 2

“…37). Some of these mutations occur in the GGF motif, which is similarly positioned in TM2 and is thought to have a structural function similar to the TXP motif of chemokine receptors (27).…”

“…The TXP motif is conserved in TM2 near the first extracellular loop of all chemokine receptors, except mouse CCR7, mouse and human CXCR5, and human XCR1 (27). Its role in CCR5 signaling was explored based on the high level of conservation and the prediction that the Pro residues disrupted the helical nature of TM2.…”

“…Although some insight into the mechanism for ligand binding has been developed, understanding of the mechanism for CCR5 activation is limited. Mutation of the Pro residue in a highly conserved sequence TXP in transmembrane domain (TM2) 2 of CCR5 has been shown to render the receptor refractory to conformational activation by ligand binding (27). In order to gain insight we have functionally expressed human CCR5 in Saccharomyces cerevisiae coupled to the pheromone response pathway by a hybrid G␣ subunit.…”

New Insights into the Mechanisms whereby Low Molecular Weight CCR5 Ligands Inhibit HIV-1 Infection

“…We found that basal activity of CCR5 was reversed to different extents by TAK779 and MVC, thus identifying both molecules as inverse agonists for the receptor. These molecules are thought to bind to a conserved hydrophobic cavity formed by the upper half of the transmembrane helices (24 -26), some of which contain amino acids previously found to be important structural determinants for activation of the receptor (55,56). So, direct or indirect interactions with these residues could explain how these, and others (29,57), small molecule CCR5 inhibitors prevent the receptor from spontaneous activation.…”

The role of CCR5 in vaccinia virus pathogenesis

“…The primary interaction occurs between the chemokine core and the exposed N-terminal domain of CCR5, then the ligand specific step is the association of the chemokine with region close to the ECL2 of CCR5 (589). Then the free N-terminus domain of the chemokine mediates receptor activation through its interaction with the TM helix bundle (70,237,495). …”