The overall structure of CXCR4 bound to the small molecule antagonist IT1t is conserved in all crystal forms with a Cα RMSD of 0.6 Å. Binding of the CVX15 cyclic antagonist peptide induced conformational differences relative to IT1t in the CXCR4-3/CVX15 structure (Cα RMSD 0.9 Å). For clarity, we focus on the highest resolution crystal form of CXCR4-2/IT1t (2.5 Å, monomer A) for discussion of the CXCR4 structural features and comparison with other GPCR structures. The final model includes 326 of the 352 residues of CXCR4 and residues 2–161 of T4L. The remaining N-terminal 26 residues did not have interpretable density and are presumed to be disordered. The main fold of CXCR4 consists of the canonical bundle of 7 TM α-helices (), which shows about the same level of structural divergence from 7TM helical bundles of previously solved GPCR structures (Cα RMSDs ~2.0–2.2 Å) (). The most striking differences in the disposition of the TM helices of CXCR4 are the following: i) The extracellular end of helix I is shifted towards the central axis of the receptor by 9 Å compared to β2AR and by more than 3 Å compared to A2AAR; ii) helix II makes a tighter helical turn at Pro922.58 resulting in ~120° rotation of its extracellular end compared to other GPCR structures (this rotation essentially introduces a one-residue gap in the sequence alignment that would result in wrong residues facing the ligand-binding pocket in a homology model that did not account for the rotation); iii) both intracellular and extracellular tips of helix IV in CXCR4 substantially deviate (~5 and ~3 Å, respectively) from their consensus positions in other GPCRs; iv) the extracellular end of helix V in CXCR4 is about one turn longer; v) helix VI has a similar shape in all structures and is characterized by a sharp kink at the highly conserved residue, Pro2546.50; however, its extracellular end is shifted by ~3 Å in CXCR4 relative to β2AR and A2AAR; and finally vi) the extracellular end of helix VII in CXCR4 is two helical turns longer than in other GPCR structures. These two extra turns place Cys2747.25 at the tip of helix VII in a strategic position to form a disulfide bond with Cys28 in the N-terminal region. Taken together, these multiple differences suggest that accurate homology modeling of even the CXCR4 TM bundle, let alone the entire structure, would be challenging.
Fig. 1 Overall fold of the CXCR4/IT1t complex and comparison with other GPCR structures. (A) Overall fold of the CXCR4-2/IT1t. The receptor is colored blue. The N-terminus, ECL1, ECL2 and ECL3 are highlighted in brown, blue, green and red, respectively. The (more ...)
The extracellular interface of CXCR4 consists of 34 N-terminal residues, extracellular loop 1 (ECL1, residues 100–104) linking helices II and III, ECL2 (residues 174–192) linking helices IV and V, and ECL3 (residues 267–273) linking helices VI and VII (). Clear density starts at Pro27, adjacent to Cys28, which pins the base of the N-terminal segment to Cys2747.25
at the tip of helix VII via a disulfide bond; these two cysteines are conserved in all chemokine receptors except CXCR5 and CXCR6 (Fig. S2
). Another disulfide links Cys1093.25
with Cys186 of ECL2, which is the largest extracellular loop in CXCR4. While ECL2 length, sequence and secondary structure vary dramatically in GPCRs, the disulfide connecting ECL2 with the extracellular end of helix III is highly conserved in chemokine receptors and most other Class A GPCRs. Both disulfide bonds at the extracellular side of CXCR4 are critical for ligand binding (29
), and the crystal structure shows that they function by constraining ECL2 and the N-terminal segment (residues 26–34), thereby shaping the entrance to the ligand binding pocket.
The intracellular side of CXCR4 contains intracellular loop 1 (ICL1, residues 65–71) linking helices I and II, ICL2 (residues 140–149) linking helices III and IV, and ICL3 (residues 225–230) linking helices V and VI, and the C-terminus. ICL3 also contains T4L inserted between Ser229 and Lys230 and flanked by short linkers (GS-T4L-GS). Structural alignment of CXCR4 with high resolution GPCR structures indicates that the intracellular half of the TM bundle is structurally more conserved (Cα RMSDs with β2
AR and rhodopsin are 1.8, 1.9 and 1.4 Å, respectively) than the extracellular half (2.6, 2.2 and 2.2 Å, respectively). Therefore, it comes as a surprise that in all five CXCR4 structures, helix VII is about one turn shorter at the intracellular side, ending right after the GPCR-conserved NPxxY motif, and that all structures lack the short α-helix VIII (). The C-terminal part of CXCR4 beyond Ala3037.54
adopts an extended conformation and participates in a number of crystal contacts with the extracellular side of a symmetry-related molecule in the highest resolution crystal form, CXCR4-2/IT1t, (Fig. S4A
), and is not traceable in the other four CXCR4 structures. Due to its structural persistence and common α-helical sequence motif (F[RK]xx[FL]xxx[LF]), helix VIII was thought to be a regular structural element of all Class A GPCRs. However, CXCR4 contains only a partially conserved motif FKxxAxxxL, and while it may be capable of forming an α-helix under certain conditions, this helix would be less stable due to replacement of Phe/Leu with Ala. In addition, CXCR4 lacks a putative palmitoylation site at the end of helix VIII, which anchors to the lipid membrane in many GPCRs.
Construct CXCR4-3 contains a T2406.36
P mutation near the intracellular side of helix VI, which results in retention of ligand binding affinity, but abolishes signaling (Table S3
and Fig. S1
). Comparison of the CXCR4-3 structure with CXCR4-1 and CXCR4-2 reveals that the only effect of the T2406.36
P mutation is the disruption of a short section of helix VI between Lys2346.30
. Since helix VI is thought to be one of the major players in the signaling mechanism (30
), disruption of its structure would likely impact G protein binding and activation. Thus, T2406.36
P represents a novel structure-based uncoupling mutation.