Abstract
Chemokines promote directional cell migration through binding to G-protein-coupled receptors, and as such are involved in a large array of developmental, homeostatic and pathological processes. They also interact with heparan sulfate (HS), the functional consequences of which depend on the respective location of the receptor- and the HS-binding sites, a detail that remains elusive for most chemokines. Here, to set up a biochemical framework to investigate how HS can regulate CXCL13 activity, we solved the solution structure of CXCL13. We showed that it comprises an unusually long and disordered C-terminal domain, appended to a classical chemokine-like structure. Using three independent experimental approaches, we found that it displays a unique association mode to HS, involving two clusters located in the α-helix and the C-terminal domain. Computational approaches were used to analyse the HS sequences preferentially recognized by the protein and gain atomic-level understanding of the CXCL13 dimerization induced upon HS binding. Starting with four sets of 254 HS tetrasaccharides, we identified 25 sequences that bind to CXCL13 monomer, among which a single one bound to CXCL13 dimer with high consistency. Importantly, we found that CXCL13 can be functionally presented to its receptor in a HS-bound form, suggesting that it can promote adhesion-dependent cell migration. Consistently, we designed CXCL13 mutations that preclude interaction with HS without affecting CXCR5-dependent cell signalling, opening the possibility to unambiguously demonstrate the role of HS in the biological function of this chemokine.
Highlights
Chemokines are a large family of soluble chemoattractant cytokines, which control the migratory patterns and the positioning of cells through interaction with G-protein-coupled receptors [1]
The summary of conformationally restricting constraints and structure quality factors are reported in table 1 for the CXCL13 monomer structure ensemble, which was deposited in the Protein Data Bank
CXCL13 includes, downstream of the a-helix, a 19 residue C-terminal extension that remains unfolded and conformationally dynamic. This C-terminal extension does not interact with the core domain of the protein, as confirmed with the analysis of a C-terminally truncated form of CXCL13 lacking the last 14 amino acids, (CXCL13-DC), which showed no disturbance of the core protein NMR signals compared with that of the full- 4 length protein
Summary
Chemokines are a large family of soluble chemoattractant cytokines, which control the migratory patterns and the positioning of cells through interaction with G-protein-coupled receptors [1]. HS is a large polysaccharide, characterized by a unique level of structural complexity. It is composed of alternating hexuronic acid, either a glucuronic acid (GlcA) or its C-5 epimer, an iduronic acid (IdoA) and an N-acetyl or N-sulfated glucosamine (GlcNAc or GlcNS). The functional importance of HS-mediated chemokine immobilization has remained difficult to evaluate: genetic manipulations targeting HS biosynthesis and structure have broad effects because they affect the interaction of HS with many other proteins; possible overlaps of the chemokine domains involved in receptor-mediated cell signalling and HS binding may obscure the interpretation of the results obtained using HS binding mutant chemokines, which would display reduced agonist capacity
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