Closing the Ring: A Fourth Extracellular Loop in Chemokine Receptors

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Science Signaling  02 Sep 2014:
Vol. 7, Issue 341, pp. pe21
DOI: 10.1126/scisignal.2005664


Chemokine receptors are heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCR) that play fundamental roles in many physiological and pathological processes. Typically, these receptors form a seven-transmembrane helix bundle, which is stabilized by a disulfide bond bridging the top of the third transmembrane segment (TM3) and the second extracellular loop (ECL2). Resolution of the three-dimensional structures of the chemokine receptors CXCR1, CXCR4, and CCR5 revealed the existence of a second disulfide bridge that links the N terminus of the receptor to the top of the seventh transmembrane segment (TM7), thereby closing the receptor into a ring. An important consequence of this second disulfide bond is the formation of an additional extracellular loop, which shapes the entrance of the ligand-binding pocket and adds rigidity to the overall surface of the receptor. Here, we discuss the features of these “pseudo-loops,” the structural requirements for their formation, and the effects they may have on receptor function.

Chemokine receptors are rhodopsin-like, heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) and are present at the surface of various cell types. By binding to their ligands, chemokine receptors regulate vital cellular mechanisms, including migration and adhesion, as well as growth and survival, but they are also involved in pathological processes such as cancer and HIV-1 infection. Previously, knowledge about the structure of chemokine receptors was built on predictions based on other class A GPCRs and on functional studies. The resolution of the three-dimensional (3D) structures of three chemokine receptors by x-ray crystallography (for CXCR4 and CCR5) and nuclear magnetic resonance (NMR) analysis (for CXCR1) has provided more precise information on the conformations adopted by members of this receptor family (Fig. 1, A to D) (13).

Fig. 1 Overall structure of the extracellular surface of chemokine receptors and the location of ECL4 pseudo-loops.

(A) Comparison between the x-ray structures of CXCR4 (blue) and CCR5 (green) showing the conserved overall arrangement of the extracellular features (ECL1, ECL2, and ECL3), as well as the location and shape of the ECL4 pseudo-loops of CXCR4 (orange) and CCR5 (red). Note that the ECL4 pseudo-loop of CXCR4 points more toward the inside of the ligand-binding pocket than does that of CCR5. The black square highlights the position of family-defining residues presented in panel F. (B) Ligand-binding pocket of CXCR4 [based on Protein Data Bank (PDB) structure 3ODU] in complex with the CXCR4 antagonist IT1t (yellow). ECL4 is colored in orange. (C) Ligand-binding pocket of CCR5 (PDB 4MBS) in complex with the small allosteric CCR5 inhibitor Maraviroc (pink). ECL4 is colored in red. (D) NMR structure of CXCR1 in a liquid crystalline phospholipid bilayer (orange) (PDB 2NLN). ECL4 is colored in red. In the absence of the small ligand, ECL2 lies on top of the ligand-binding pocket, blocking its access, and TM1 is off-centered from the TM circle in comparison with ECL2 of CXCR4 and CCR5. (E) Arrangement and position of CRS1 and CRS2 in CXCR4. The N terminus, ECL2, and ECL4 are colored in gray, green, and orange, respectively. The complete structure of CXCR4 was generated by molecular dynamic simulation after engraftment of the 28 N-terminal residues to the resolved x-ray structure (PDB: 3ODU) (4). (F) Positional conservation of residues at positions C+6 in CCR5 (family A, red) and C+4 in CXCR4 (family B, orange). (G) Sequences and classification of chemokine receptor ECL4 pseudo-loops. The ECL4 pseudo-loop of chemokine receptors encompasses residues from the cysteine (C), involved in a disulfide bridge with the top of TM7 (at the end of ECL3), to the first amino acid residue of TM1. ECL4 classification is based on the presence of two conserved positively charged residues at positions C+2 and C+6 (family A) or a negatively charged residue at positions C+3 or C+4 (family B). ECL4s containing both signatures are classified as family A/B, whereas those presenting none of the conserved residues are classified as family C. Residues belonging to incomplete signatures are underlined. The asterisk indicates that no cysteine is present in the N terminus of CXCR6.


Similar to other rhodopsin-like GPCRs, chemokine receptors consist of a flexible extracellular N terminus that is followed by a bundle of seven hydrophobic plasma membrane–spanning α helices [known as transmembrane (TM) domains] that are connected by three hydrophilic extracellular loops (ECLs) and three intracellular loops (ICLs). In addition to the canonical disulfide bond that links the top of the third TM domain (TM3, at the end of ECL1) to the middle of ECL2, all three currently available 3D structures of chemokine receptors demonstrate the presence of a second disulfide bridge between the N terminus of the receptor and the top of TM7, at the end of ECL3 (Fig. 1). As a consequence, the C-terminal residues of the N terminus of the receptor form an extracellular loop (which is termed “ECL4”), which connects TM1 and TM7 and closes the receptor into a ring-like conformation (Fig. 1A). This fourth loop consists of six (for CCR5) or eight amino acid residues (for CXCR1 and CXCR4) (Fig. 1G) and is thus comparable to ECL1 and ECL3, which contain between four and eight residues each. With the exception of CXCR6, all of the chemokine receptors have a cysteine in the last third of their N-terminal regions, which suggests that the additional disulfide bridge is conserved. Although the formation of this disulfide bridge is critical for the function of several chemokine receptors, the role of the additional loop in ligand recognition and receptor activation has been given less attention.

The formation of ECL4 at the surface of chemokine receptors requires structural adaptations and possibly has consequences on receptor function. In CXCR1, CXCR4, and CCR5, the transmembrane helix that forms TM7 is two turns longer than that in other GPCRs. Elongation of the chemokine receptor helix seems to be required to position the conserved cysteine toward the inner face of TM7, which favors its engagement in the disulfide bridge with the N terminus of the receptor (Fig. 1, A and E). The ECL4 pseudo-loop may play an important role in chemokine recognition. Chemokine receptors are thought to bind to their ligands through a two-step mechanism that involves successive interactions between the chemokine and both the flexible N terminus of the receptor [chemokine recognition site 1 (CRS1)] and a pocket located in the vicinity of the transmembrane segments and the extracellular loops (CRS2) (4, 5). Note that ECL4 and the disulfide bond between the N terminus and TM7 reposition the remaining flexible part of the N-terminal region of the receptor from the top of TM1 to the top of TM7 alongside ECL3 (Fig. 1E). Such a delocalization is likely necessary for chemokine binding and would provide an optimal orientation of the flexible N terminus of the receptor (CRS1) with respect to CRS2. This repositioning may be further facilitated by the proline residue that often directly precedes the conserved cysteine, which forms a kink in CRS1 and brings CRS1 in front of the β hairpin of ECL2, a major determinant of CRS2 in CXCR4 (Fig. 1, A and E) (6). ECL4 also influences the shape, size, and charge of the entrance of the transmembrane binding pocket for endogenous ligands (CRS2) and small pharmacological modulators (Fig. 1, B to D). Similar to the canonical disulfide bridge between TM3 and ECL2, the bond between the N terminus and TM7 may also contribute to the overall stability and rigidity of the receptor, as well as to the conformational changes that occur upon chemokine binding. Finally, this loop may limit the diffusion of small molecules across the helix bundle, and it may participate in receptor-receptor interactions, type I dimerization, or both.

Despite difficulties in predicting the starting residue of TM1, and although there is little ECL4 sequence similarity among receptors, we identified three subfamilies of chemokine receptors that are characterized by different molecular signatures within their pseudo-loops (Fig. 1G) (4, 7). The receptors CCR1, CCR5, and CCR9 share conserved, positively charged residues at positions C+2 and C+6 (family A), whereas CCR6, CCR8, CCR10, all of the CXC receptors, CX3CR1, and the atypical chemokine receptor ACKR2 (D6) have a negatively charged residue at position C+3 or C+4 (family B). The side chains of the residues that define family A (Lys26, C+6) and family B (Glu32, C+4) are well aligned in the superposed x-ray structures of CXCR4 and CCR5 (Fig. 1F) and point toward the inner face of the receptors, suggesting that this position may be of importance for receptor function. This observation is consistent with data demonstrating that the Asp25 of CX3CR1 (C+4) is critical for binding to its ligand CX3CL1 (fractalkine) (8) and that Glu32 of CXCR4 (C+4) is predicted to interact with the N-terminal lysine of CXCL12 (also known as SDF1-α), which accounts for its agonist activity (9). Other receptors, such as CCR2, CCR3, CCR4, CCR7, and ACKR4 (CCX-CKR) bear both types of signatures (family A/B). The receptors XCR1, ACKR1 (DARC), and ACKR3 (CXCR7) display no feature that enables their classification into one of the two families (family C). In contrast to sequences preceding the conserved cysteine, no posttranslational modifications are predicted among the different ECL4s, except for that of ACKR3, which displays a putative N-glycosylation site (NKS) at position C+5.

The presence of a fourth ECL and its molecular signatures may not be restricted to chemokine receptors. Indeed, the additional cysteine residues in the N terminus and TM7 (ECL3) are also found in ~30% of receptors that belong to the rhodopsin family, including receptors for lysophospholipid (LPA), bradykinin (B1-2), endothelin (ETA-B), melanocortin (MC1-5), serotonin (5-HT), purinergic (P2Y), and orphan receptors. The structure of the latest resolved rhodopsin-like receptor, P2Y12, revealed the presence of a pseudo-loop equivalent to that found in the chemokine receptors (10); however, the conservation of these residues does not necessarily imply the formation of a pseudo-loop, as is shown by the structures of the dopamine receptor D3 and the serotonin receptor 5-HT1B, which lack a disulfide bridge between the two conserved cysteines (11, 12). Therefore, in the near future, the presence and the exact role of ECL4s in ligand-binding, signal transduction, and receptor interactions will need to be addressed in more detail, not only for chemokine receptors, but also for other receptor families.

References and Notes

Acknowledgments: The authors thank C. Seguin-Devaux, J. Hanson, and P. Lambert for fruitful discussions and critical reading of the manuscript and K. Arumugam for molecular modeling. Funding: This manuscript was supported by the Centre de Recherche Public de la Santé (CRP-Santé), Luxembourg, grant 20100708; the Fonds National de la Recherche, Luxembourg, grants AFR-3004509 and CORE C11/BM/1209287; and Télévie, grant 7.4568.14.
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