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Abstract
Glycosaminoglycans (GAGs) bind a large array of proteins and mediate fundamental and diverse roles in human physiology. Ion pair interactions between protein lysines/arginines and GAG sulfates/carboxylates mediate binding. Neutrophil-activating chemokines (NAC) are GAG-binding proteins, and their sequences reveal high selectivity for lysines over arginines indicating they are functionally not equivalent. NAC binding to GAGs impacts gradient formation, receptor functions, and endothelial activation, which together regulate different components of neutrophil migration. We characterized the consequence of mutating lysine to arginine in NAC CXCL8, a well-characterized GAG-binding protein. We chose three lysines — two highly conserved lysines (K20 and K64) and a CXCL8-specific lysine (K67). Interestingly, the double K64R/K20R and K64R/K67R mutants are highly impaired in recruiting neutrophils in a mouse model. Further, both the mutants bind GAG heparin with higher affinity but show similar receptor activity. NMR and MD studies indicate that the structures are essentially identical to the WT, but the mutations alter the network of intramolecular ion pair interactions. These observations collectively indicate that the reduced in vivo recruitment is due to altered GAG interactions, higher GAG binding affinity can be detrimental, and specificity of lysines fine-tunes in vivo GAG interactions and function.
Highlights
Glycosaminoglycans (GAGs) are polysaccharides that are ubiquitous components of cell surfaces and the extracellular matrix[1,2,3,4]
Our NMR-based model of CXCL8-heparin octasaccharide shows that K64 lies in the middle of the binding surface suggesting it functions as an important anchor point, and mutating this lysine to alanine significantly impairs neutrophil recruitment[32]
We designed three mutants centered on K64 to address how conserved and chemokine-specific lysines influence structure and function — K64R mutant, a double K20R/ K64R mutant that addresses the role of highly conserved lysines and a double K64R/K67R mutant that addresses the role of a highly conserved and CXCL8-specific lysine
Summary
Glycosaminoglycans (GAGs) are polysaccharides that are ubiquitous components of cell surfaces and the extracellular matrix[1,2,3,4] They bind hundreds of proteins including chemokines and growth factors, and mediate and regulate diverse fundamental roles in human pathophysiology from inflammation and organogenesis to cancer progression[5,6,7,8,9]. In a recent MD study of a protein-DNA complex, it has been shown that the mean lifetimes of direct H-bond/ion pair interactions of arginine are much longer and less dynamic than those involving lysines[26]. The conserved arginine (B1) is involved in GAG interactions only in CXCL1, but is a very critical residue for receptor activation for all seven chemokines, indicating that the major function of this conserved arginine is not for GAG binding. The preference for lysines stands out, suggesting that the structural features of lysine provide specificity that cannot be conferred by an arginine
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