Structure of CC Chemokine Receptor 5 with a Potent Chemokine Antagonist Reveals Mechanisms of Chemokine Recognition and Molecular Mimicry by HIV

Abstract

CCR5 is the primary chemokine receptor utilized by HIV to infect leukocytes, whereas CCR5 ligands inhibit infection by blocking CCR5 engagement with HIV gp120. To guide the design of improved therapeutics, we solved the structure of CCR5 in complex with chemokine antagonist [5P7]CCL5. Several structural features appeared to contribute to the anti-HIV potency of [5P7]CCL5, including the distinct chemokine orientation relative to the receptor, the near-complete occupancy of the receptor binding pocket, the dense network of intermolecular hydrogen bonds, and the similarity of binding determinants with the FDA-approved HIV inhibitor Maraviroc. Molecular modeling indicated that HIV gp120 mimicked the chemokine interaction with CCR5, providing an explanation for the ability of CCR5 to recognize diverse ligands and gp120 variants. Our findings reveal that structural plasticity facilitates receptor-chemokine specificity and enables exploitation by HIV, and provide insight into the design of small molecule and protein inhibitors for HIV and other CCR5-mediated diseases.

Keywords: CCL5/RANTES; CCR5-gp120 interaction; Chemokine antagonist; G protein-coupled receptor (GPCR); HIV entry inhibitor; V3 loop; maraviroc; membrane protein structure; two-site model; viral mimicry.

Figure 1. The structure of the complex between CCR5 and the anti-HIV chemokine variant [5P7]CCL5

(A) Structure of [5P7]CCL5-bound CCR5, viewed parallel to the membrane plane. (B-C) Structure viewed from the extracellular (B) and intracellular (C) side. Loops connecting the TM helices are lime green. Residues 0–4 of the chemokine are shown as sticks, with the electron density of the Fo-Fc omit map contoured at 3σ. (D) View of [5P7]CCL5 with residues 0–9 and the 30s loop residues shown as sticks. The electron density of the Fo-Fc omit maps is shown and contoured at 3σ for the engineered N-terminus (gray mesh) and 30s loop (green mesh), respectively. See also Figure S1 and Table S1.

Figure 2. The CCR5-[5P7]CCL5 complex shows additional, distinct interaction epitopes in the context of the conserved two-site architecture

(A) Three epitopes observed in earlier receptor-chemokine structures are highlighted on the structure of CCR5-[5P7]CCL5: CRS1 (purple), CRS1.5 (gold), and CRS2 (cyan). (B) Comparison of the CRS1.5 interaction geometry and hydrogen bonding pattern between CCR5-[5P7]CCL5 and earlier receptor-chemokine structures. Chemokines are superimposed by their globular cores and shown as molecular surfaces on the left and ribbons on the right. The fragments involving the N-terminus and helix I of each receptor are shown as ribbons with the conserved Pro of the 19-PC-20 motif in sticks. The Pro invariably packs against the conserved disulfide (yellow surface on left and yellow sticks on right) of the chemokine, and the flanking residues form invariable backbone hydrogen bonds to the chemokine proximal N-terminus and β₃-strand. (C) The diverse positions of the chemokine globular cores with respect to the TM domains of the receptors are shown for CCR5-[5P7]CCL5, US28-CX3CL1 and CXCR4-vMIP-II. The TM domains are superimposed and shown in ribbons, chemokines are shown as molecular surfaces and contoured for clarity. The C-terminal helix and Cα atom of the central residue of the β-sheet (V40 in CCL5) of each chemokine are shown as a cylinder and a sphere, respectively. (D-F) The interactions formed by the [5P7]CCL5 β₁-strand with ECL2 of CCR5 (green) and its 30s loop with CCR5 ECL3 and helices V-VII deep within the CCR5 binding pocket (red), are highlighted and compared with similar regions of US28-CX3CL1 and CXCR4-vMIP-II in (E and F). The β₁-strand interaction is virtually absent from both US28-CX3CL1 (E) and CXCR4-vMIPII (F) complexes. The chemokine 30s loop contacts ECL2 in the US28-CX3CL1 complex (E) but shows almost no interaction in the CXCR4-vMIPII complex (F). See also Figure S2.

Figure 3. Structural determinants of [5P7]CCL5 affinity in CRS2, and overlap with the HIV inhibitor Maraviroc

(A-C) Steric packing of the chemokine N-terminus (ribbon and sticks), in the TM domain pocket of the receptor (cut-away surfaces colored by residue sidechain properties). (A) and (C) are views along the plane of the membrane ((C) is designated as “front”, (A) is “right”). (B) shows a view across the plane of the membrane from the extracellular side. Prominent non-polar subpockets mentioned in the text are highlighted with yellow contours. (D-E) Steric packing of the small molecule antagonist Maraviroc in complex with CCR5 is shown with views and orientations identical to those in (B) and (C). (F-G) Polar interactions of residues 0–4 (F) and residues 0–9 (G) of [5P7]CCL5 with CCR5. Direct and water-mediated hydrogen bonds are shown in black and cyan, respectively. (F) presents a view across the plane of the membrane from the extracellular side, identical to (B) and (D). (G) is a view along the plane of the membrane from the “left”. See also Figure S3, Table S2, Table S3 and Table S4.

Figure 4. Water molecules in the TM domain of the CCR5-[5P7]CCL5 structure compared to other GPCR complexes

Within the TM domain, the water-mediated network is shown traversing from the antagonist binding site (dark blue ribbon/sticks), through the allosteric sodium site (purple spheres), to the hydrophobic layer (matte surface) at the intracellular end of the receptors. Water is shown as red sticks overlaid on magenta spheres. (A) Water network in the CCR5-[5P7]CCL5 structure. The figure shows that despite the missing sodium ion in the CCR5 structure (possibly due to incompatibility with residues G^3.35 and G^3.39), the overall polar network is maintained. (B) In the adenosine A_2A receptor (PDB ID: 4EIY), residues D^2.50 and S^3.39 coordinate a transmembrane sodium ion along with three water molecules (orange). (C) In the δ-opioid receptor (PDB ID: 4N6H), residues D^2.50, N^3.35, S^3.39, and two water molecules (orange) participate in sodium coordination.

Figure 5. N-loop and 30s loop interactions represent new motifs in receptor-chemokine interactions

(A) Steric interactions of the N-loop (brown), 30s loop (red), and β₁-strand (green) of [5P7]CCL5 (blue ribbon except for the highlighted parts) with the TM domain and ECLs of CCR5. Residues involved in aromatic stacking interactions are shown as spheres. The receptor is shown as a cut-away surface colored as in Figure 3. (B) Polar interactions of [5P7]CCL5 30s loop (red), and β₁-strand (green) with the CCR5 binding pocket and ECLs. Direct and water-mediated receptor-chemokine hydrogen bonds are shown in black and cyan, respectively. (C) The effect of mutations of [5P7]CCL5 residues K33 and F12 on the thermostability of CCR5-[5P7]CCL5 complexes. See also Table S2 and Table S3.

Figure 6. Modeling provides insights into CCR5 interaction with its endogenous chemokine agonist CCL5

(A) Overall view of the CCR5-CCL5 model. Residues 1–9 that differ between CCL5 and [5P7]CCL5 are highlighted in saddle brown. Tyrosine sulfates on the receptor N-terminus are shown as spheres. Residues important for the CRS1 interaction (by mutagenesis) are labeled and highlighted in color. (B) Predicted polar interactions in CRS1. (C-E) Predicted steric packing of the CCL5 N-terminus (ribbon and sticks) in the TM domain pocket of the CCR5 (cut-away surfaces colored by residue sidechain properties, as in Figure 3). (C) and (E) are two views along the plane of the membrane, “right” and “front”, respectively, as in Figure 3. (D) shows a view across the plane of the membrane from the extracellular side. (F-G) Predicted polar interactions of the distal N-terminus (residues 1-3, F) and the complete N-terminus (residues 1-9, G) of CCL5 with receptor residues. Direct and water-mediated receptor-chemokine hydrogen bonds are shown in black and cyan, respectively. (F) presents a view identical to (D). (G) is the “front” view, identical to (E). See also Figure S4, Table S3 and Table S4.

Figure 7. Insights into CCR5 interaction with HIV gp120 and inhibition of this interaction by chemokines

(A) Ensemble of models of complexes between CCR5 and a fragment of HIV gp120 containing its V3 loop and C4 region. The ensemble illustrates multiple possible orientations of gp120 with respect to the receptor, enabled via the inherent flexibility of the interaction interface. (B) Representative model of CCR5 (ribbon) in complex with the V3/C4 fragment of HIV gp120 (strain KNH1135, molecular surface). Interaction sites for the receptor N-terminus, the conserved 19-PC-20 motif, and the TM binding pocket are highlighted to illustrate architectural similarity to complexes with chemokines. (C) Same model as in (B), showing interactions of important residues. CCR5 is shown in ribbon and a cut-away molecular surface, gp120 in ribbon surrounded by a transparent simplified molecular surface. Residues mentioned in the text are shown in sticks. Residue P311 in the conserved GPG[RQ] motif of the gp120 V3 crown, as well as the CCR5 N-terminal tyrosine sulfates are shown as spheres. (D) Prior mutagenesis of gp120 mapped onto the surface of the gp120 V3/C4 fragment using the same model as in (B). Residues that affect binding of a sulfo-tyrosinated CCR5 N-terminal peptide (2–18) are colored in magenta. Residues whose mutations show effects only in the context of full-length CCR5 are show in cyan. The predicted architecture and topology of the complex are highly consistent with mutagenesis. (E-F) Among chemokine crystallized with receptors, [5P7]CCL5 is unique in its ability to entirely fill the binding pocket of CCR5 (E). By contrast, binding of CX3CL1 to US28 (F) or vMIPII to CXCR4 (G) leads to only partial occupancy of the pocket, with much of the major subpocket remaining accessible (yellow contours). Receptors are shown as cut-away surfaces, chemokines as simplified cut-away surfaces and ribbons. See also Figure S5, Table S5 and Table S6.