CXCL1/MGSA Is a Novel Glycosaminoglycan (GAG)-binding Chemokine: STRUCTURAL EVIDENCE FOR TWO DISTINCT NON-OVERLAPPING BINDING DOMAINS

Krishna Mohan Sepuru,Krishna Rajarathnam

doi:10.1074/jbc.m115.697888

Krishna Mohan Sepuru, Krishna Rajarathnam

Open Access

https://doi.org/10.1074/jbc.m115.697888

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Abstract

In humans, the chemokine CXCL1/MGSA (hCXCL1) plays fundamental and diverse roles in pathophysiology, from microbial killing to cancer progression, by orchestrating the directed migration of immune and non-immune cells. Cellular trafficking is highly regulated and requires concentration gradients that are achieved by interactions with sulfated glycosaminoglycans (GAGs). However, very little is known regarding the structural basis underlying hCXCL1-GAG interactions. We addressed this by characterizing the binding of GAG heparin oligosaccharides to hCXCL1 using NMR spectroscopy. Binding experiments under conditions at which hCXCL1 exists as monomers and dimers indicate that the dimer is the high-affinity GAG ligand. NMR experiments and modeling studies indicate that lysine and arginine residues mediate binding and that they are located in two non-overlapping domains. One domain, consisting of N-loop and C-helical residues (defined as α-domain) has also been identified previously as the GAG-binding domain for the related chemokine CXCL8/IL-8. The second domain, consisting of residues from the N terminus, 40s turn, and third β-strand (defined as β-domain) is novel. Eliminating β-domain binding by mutagenesis does not perturb α-domain binding, indicating two independent GAG-binding sites. It is known that N-loop and N-terminal residues mediate receptor activation, and we show that these residues are also involved in extensive GAG interactions. We also show that the GAG-bound hCXCL1 completely occlude receptor binding. We conclude that hCXCL1-GAG interactions provide stringent control over regulating chemokine levels and receptor accessibility and activation, and that chemotactic gradients mediate cellular trafficking to the target site.

Highlights

Chemokines, a large family of small soluble proteins, are highly versatile and play fundamental roles in diverse functions, from combating infection and initiating tissue repair to regulating metabolism and organ development [1, 2]
Our NMR studies indicate that the molecular basis of GAG heparin binding to human CXCL1 (hCXCL1) is novel and that it is strikingly different from the closely related ELR chemokine CXCL8
We have shown recently that heparin-binding residues in CXCL8 can be classified as core and peripheral residues and that GAG can bind in different geometries [26]

Summary

Introduction

Chemokines, a large family of small soluble proteins, are highly versatile and play fundamental roles in diverse functions, from combating infection and initiating tissue repair to regulating metabolism and organ development [1, 2]. Common to these various functions is the directed movement of various cell types to distal and remote locations. One of the domains has been observed for the related chemokine CXCL8/ IL-8, the presence of a second domain is novel Residues from both GAG-binding domains are involved in receptor interactions, indicating that GAG-bound hCXCL1 cannot activate the receptor. We propose that two independent GAG binding domains impart better control over fine-tuning chemokine concentration gradients and receptor activation for orchestrated cellular trafficking to the target site

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