Not surprisingly, P32A showed overall higher activity than the P32G mutant. could not be correlated with the specific mutation, receptor affinity or subtype, or a specific signalling pathway. NMR studies indicated that the GP motif does not influence Site-I interactions, but molecular dynamics simulations suggested that this motif dictates substates of the CXCL8 conformational LY2794193 ensemble. We conclude that the GP motif enables diverse receptor functions by controlling cross-talk between Site-I and Site-II, and further propose that LY2794193 the repertoire of chemokine functions is best described by a conformational ensemble model in which a network of long-range coupled indirect interactions mediate receptor activity. Keywords:chemokine, conformational ensemble, G-protein-coupled receptor, long-range coupling, signalling == INTRODUCTION == Chemokines, a large family of ~810 kDa proteins, play fundamental roles from developmental biology to trafficking immune cells and combating infection by activating receptors that belong to the GPCR (G-protein-coupled receptor) class [1]. They show a wide repertoire of receptor functions, as a given chemokine can bind single or multiple receptor(s), and binding to any one receptor can elicit multiple signalling pathways and different activities [1,2]. According to the prevalent model, chemokine receptor activation involves interactions between chemokine N-loop and receptor N-terminal residues (Site-I), and between chemokine N-terminal and receptor extracellular/transmembrane residues (Site-II) [26]. This model, although appealing in its simplicity, fails to describe the large variation in receptor selectivity, affinity and activity for a given chemokine and among chemokines. Sequence analysis of neutrophil-activating chemokines in humans and across other species show that the GP (glycine-proline) residues which form a -turn in the 30s-loop are highly conserved (Figure 1A). Structures reveal that the 30s-loop is located distal to both the N-terminal and N-loop regions (Figure 1B). High conservation implies that it must play some crucial role for structure and/or function. Therefore the GP motif could be essential for the structural fold and/or for receptor activity by directly binding receptors or indirectly influencing how N-terminal/N-loop residues bind and activate the receptors. == Figure 1. Structural characteristics of CXCL8. == (A) Sequence alignment of human CXCL8 (hCXCL8) and related neutrophil-activating chemokines, and CXCL8 from other species. N-terminal ELR residues (in green) are absolutely conserved and the 30s-loop GP motif (in red) is highly conserved, except in CXCL7. The N-loop residues are indicated by a blue bar. (B) Tertiary structural components of the CXCL8 monomer. The 30s-loop, N-loop (Site-I), ELR LY2794193 residues (Site-II) and disulfide bonds (black) are highlighted. The GP motif in the -turn of the 30s-loop are shown as sticks, and the hydrogen bonds between Gly31and Cys34are shown as broken lines (see insert). Chemokine CXCL8 [also known as IL-8 (interleukin-8)] recruits neutrophils by LY2794193 activating the CXCR1 and CXCR2 receptors. Structurefunction studies have shown that the N-terminal ELR and N-loop residues play important roles in defining affinity and receptor activity [5,7,8]. Chemokine PF-4/CXCL4 has the GP motif and is not active for CXCR1/CXCR2 receptors, but becomes active on introducing the N-terminal ELR motif [9]. The chemokine IP-10/CXCL10 does not have the GP motif and is not active against CXCR1/CXCR2 receptors [10]. However, a CXCL10/CXCL8 chimaera containing the 30s-loop, N-terminal ELR and N-loop residues of CXCL8 is active, but a chimaera containing the 30s-loop and ELR residues is not active [7]. Therefore the mere presence of 30s-loop GP motif or even the additional presence of ELR residues are not sufficient for CXCL8 activity, indicating that receptor activation is finely tuned and requires optimal cross-talk among N-terminal, N-loop and 30s-loop residues. To address the role of the GP motif in CXCL8 function, we have now characterized the structural and functional properties of a panel of GP mutants (G31A, G31V, P32G and P32A). The mutants show large differences in activity that could not be correlated with the nature of the mutation, receptor type and/or a specific signalling pathway. NMR studies show that the mutations do not perturb the global fold, any local structural change could not be correlated with functional changes, and that they bind the receptor N-domain like WT (wild-type) indicating that the GP motif does not participate or influence Site-I interactions. MD (molecular dynamics) simulations show that perturbing the GP motif leads to conformational rearrangement and altered dynamics resulting in access to non-native conformations, thus providing a rationale for the stringent requirement of the GP motif for native function. We propose that the GP motif functions LY2794193 as a conformational switch and dictates the distribution and selection of the functionally competent EDNRA conformational substates that activate various signalling pathways. We further propose that the conformational selection model can best describe the spectrum of chemokine receptor functions [11,12]. == MATERIALS AND METHODS == == Cloning, expression and purification of CXCL8 GP mutants == The 30s-loop G31A,.