Dataset Information

Functional anatomy of the full-length CXCR4-CXCL12 complex systematically dissected by quantitative model-guided mutagenesis.

ABSTRACT: Because of their prominent roles in development, cancer, and HIV, the chemokine receptor CXCR4 and its ligand CXCL12 have been the subject of numerous structural and functional studies, but the determinants of ligand binding, selectivity, and signaling are still poorly understood. Here, building on our latest structural model, we used a systematic mutagenesis strategy to dissect the functional anatomy of the CXCR4-CXCL12 complex. Key charge swap mutagenesis experiments provided evidence for pairwise interactions between oppositely charged residues in the receptor and chemokine, confirming the accuracy of the predicted orientation of the chemokine relative to the receptor and providing insight into ligand selectivity. Progressive deletion of N-terminal residues revealed an unexpected contribution of the receptor N terminus to chemokine signaling. This finding challenges a longstanding "two-site" hypothesis about the essential features of the receptor-chemokine interaction in which the N terminus contributes only to binding affinity. Our results suggest that although the interaction of the chemokine N terminus with the receptor-binding pocket is the key driver of signaling, the signaling amplitude depends on the extent to which the receptor N terminus binds the chemokine. Together with systematic characterization of other epitopes, these data enable us to propose an experimentally consistent structural model for how CXCL12 binds CXCR4 and initiates signal transmission through the receptor transmembrane domain.

SUBMITTER: Stephens BS

PROVIDER: S-EPMC7437921 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Functional anatomy of the full-length CXCR4-CXCL12 complex systematically dissected by quantitative model-guided mutagenesis.

Stephens Bryan S BS Ngo Tony T Kufareva Irina I Handel Tracy M TM

Science signaling 20200714 640

Because of their prominent roles in development, cancer, and HIV, the chemokine receptor CXCR4 and its ligand CXCL12 have been the subject of numerous structural and functional studies, but the determinants of ligand binding, selectivity, and signaling are still poorly understood. Here, building on our latest structural model, we used a systematic mutagenesis strategy to dissect the functional anatomy of the CXCR4-CXCL12 complex. Key charge swap mutagenesis experiments provided evidence for pair ...[more]

PMID: 32665413

Similar Datasets

Project description:Cranial neural crest cells (CNCCs), identified by expression of transcription factor Sox9, migrate to the first branchial arch and undergo proliferation and differentiation to form the cartilage and bone structures of the orofacial region, including the palatal bone. Sox9 promotes osteogenic differentiation and stimulates CXCL12-CXCR4 chemokine-receptor signaling, which elevates alkaline phosphatase (ALP)-activity in osteoblasts to initiate bone mineralization. Disintegration of the midline epithelial seam (MES) is crucial for palatal fusion. Since we earlier demonstrated chemokine-receptor mediated signaling by the MES, we hypothesized that chemokine CXCL12 is expressed by the disintegrating MES to promote the formation of an osteogenic center by CXCR4-positive osteoblasts. Disturbed migration of CNCCs by excess oxidative and inflammatory stress is associated with increased risk of cleft lip and palate (CLP). The cytoprotective heme oxygenase (HO) enzymes are powerful guardians harnessing injurious oxidative and inflammatory stressors and enhances osteogenic ALP-activity. By contrast, abrogation of HO-1 or HO-2 expression promotes pregnancy pathologies. We postulate that Sox9, CXCR4, and HO-1 are expressed in the ALP-activity positive osteogenic regions within the CNCCs-derived palatal mesenchyme. To investigate these hypotheses, we studied expression of Sox9, CXCL12, CXCR4, and HO-1 in relation to palatal osteogenesis between E15 and E16 using (immuno)histochemical staining of coronal palatal sections in wild-type (wt) mice. In addition, the effects of abrogated HO-2 expression in HO-2 KO mice and inhibited HO-1 and HO-2 activity by administrating HO-enzyme activity inhibitor SnMP at E11 in wt mice were investigated at E15 or E16 following palatal fusion. Overexpression of Sox9, CXCL12, CXCR4, and HO-1 was detected in the ALP-activity positive osteogenic regions within the palatal mesenchyme. Overexpression of Sox9 and CXCL12 by the disintegrating MES was detected. Neither palatal fusion nor MES disintegration seemed affected by either HO-2 abrogation or inhibition of HO-activity. Sox9 progenitors seem important to maintain the CXCR4-positive osteoblast pool to drive osteogenesis. Sox9 expression may facilitate MES disintegration and palatal fusion by promoting epithelial-to-mesenchymal transformation (EMT). CXCL12 expression by the MES and the palatal mesenchyme may promote osteogenic differentiation to create osteogenic centers. This study provides novel evidence that CXCL12-CXCR4 interplay facilitates palatal osteogenesis and palatal fusion in mice.

Project description:The chemokine CXCL12 and its G protein-coupled receptor (GPCR) CXCR4 are high-priority clinical targets because of their involvement in metastatic cancers (also implicated in autoimmune disease and cardiovascular disease). Because chemokines interact with two distinct sites to bind and activate their receptors, both the GPCRs and chemokines are potential targets for small molecule inhibition. A number of chemokines have been validated as targets for drug development, but virtually all drug discovery efforts focus on the GPCRs. However, all CXCR4 receptor antagonists with the exception of MSX-122 have failed in clinical trials due to unmanageable toxicities, emphasizing the need for alternative strategies to interfere with CXCL12/CXCR4-guided metastatic homing. Although targeting the relatively featureless surface of CXCL12 was presumed to be challenging, focusing efforts at the sulfotyrosine (sY) binding pockets proved successful for procuring initial hits. Using a hybrid structure-based in silico/NMR screening strategy, we recently identified a ligand that occludes the receptor recognition site. From this initial hit, we designed a small fragment library containing only nine tetrazole derivatives using a fragment-based and bioisostere approach to target the sY binding sites of CXCL12. Compound binding modes and affinities were studied by 2D NMR spectroscopy, X-ray crystallography, molecular docking and cell-based functional assays. Our results demonstrate that the sY binding sites are conducive to the development of high affinity inhibitors with better ligand efficiency (LE) than typical protein-protein interaction inhibitors (LE ? 0.24). Our novel tetrazole-based fragment 18 was identified to bind the sY21 site with a K(d) of 24 ?M (LE = 0.30). Optimization of 18 yielded compound 25 which specifically inhibits CXCL12-induced migration with an improvement in potency over the initial hit 9. The fragment from this library that exhibited the highest affinity and ligand efficiency (11: K(d) = 13 ?M, LE = 0.33) may serve as a starting point for development of inhibitors targeting the sY12 site.

Dataset Information

Functional anatomy of the full-length CXCR4-CXCL12 complex systematically dissected by quantitative model-guided mutagenesis.

Publications

Functional anatomy of the full-length CXCR4-CXCL12 complex systematically dissected by quantitative model-guided mutagenesis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure