Solution structure of CXCL13 and heparan sulfate binding show that GAG binding site and cellular signalling rely on distinct domains.

Yoan R. Monneau,Romain R. Vivès,Balaji Nagarajan,Françoise Baleux,Nehru Viji Sankaranarayanan,Umesh R. Desai,Fernando Arenzana-Seidedos,Hugues Lortat-Jacob,Lingjie Luo

doi:10.1098/rsob.170133

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

Introduction

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

Methods

Results

Conclusion