Kirti V. Sawant

Molecular Basis of Glycosaminoglycan Heparin Binding to the Chemokine CXCL1 Dimer

Background: Glycosaminoglycan (GAG)-chemokine dimer interactions regulate neutrophil trafficking, but the molecular basis underlying their interactions is not well understood. Results: NMR studies of murine CXCL1 indicate that heparin spans the dimer interface and enhances its structural integrity and stability. Conclusion: Heparin binding modulates multiple structural properties of the chemokine dimer. Significance: This study provides novel structural insights into how chemokine dimers orchestrate neutrophil recruitment. Glycosaminoglycan (GAG)-bound and soluble chemokine gradients in the vasculature and extracellular matrix mediate neutrophil recruitment to the site of microbial infection and sterile injury in the host tissue. However, the molecular principles by which chemokine-GAG interactions orchestrate these gradients are poorly understood. This, in part, can be directly attributed to the complex interrelationship between the chemokine monomer-dimer equilibrium and binding geometry and affinities that are also intimately linked to GAG length. To address some of this missing knowledge, we have characterized the structural basis of heparin binding to the murine CXCL1 dimer. CXCL1 is a neutrophil-activating chemokine and exists as both monomers and dimers (Kd = 36 μm). To avoid interference from monomer-GAG interactions, we designed a trapped dimer (dCXCL1) by introducing a disulfide bridge across the dimer interface. We characterized the binding of GAG heparin octasaccharide to dCXCL1 using solution NMR spectroscopy. Our studies show that octasaccharide binds orthogonally to the interhelical axis and spans the dimer interface and that heparin binding enhances the structural integrity of the C-terminal helical residues and stability of the dimer. We generated a quadruple mutant (H20A/K22A/K62A/K66A) on the basis of the binding data and observed that this mutant failed to bind heparin octasaccharide, validating our structural model. We propose that the stability enhancement of dimers upon GAG binding regulates in vivo neutrophil trafficking by increasing the lifetime of “active” chemokines, and that this structural knowledge could be exploited for designing inhibitors that disrupt chemokine-GAG interactions and neutrophil homing to the target tissue.

Chemokine CXCL1 dimer is a potent agonist for the CXCR2 receptor

Background: Chemokines exist reversibly as monomers and dimers, but dimer activity remains poorly defined. Results: A disulfide-linked CXCL1 dimer is highly active, and NMR studies show that dimer binds CXCR2 like the monomer. Conclusion: The potent activity of CXCL1 dimer is novel. Significance: Chemokine dimers can be highly active to completely inactive, indicating that dimerization fine-tunes chemokine-specific in vivo functions. The CXCL1/CXCR2 axis plays a crucial role in recruiting neutrophils in response to microbial infection and tissue injury, and dysfunction in this process has been implicated in various inflammatory diseases. Chemokines exist as monomers and dimers, and compelling evidence now exists that both forms regulate in vivo function. Therefore, knowledge of the receptor activities of both CXCL1 monomer and dimer is essential to describe the molecular mechanisms by which they orchestrate neutrophil function. The monomer-dimer equilibrium constant (∼20 μm) and the CXCR2 binding constant (1 nm) indicate that WT CXCL1 is active as a monomer. To characterize dimer activity, we generated a trapped dimer by introducing a disulfide across the dimer interface. This disulfide-linked CXCL1 dimer binds CXCR2 with nanomolar affinity and shows potent agonist activity in various cellular assays. We also compared the receptor binding mechanism of this dimer with that of a CXCL1 monomer, generated by deleting the C-terminal residues that stabilize the dimer interface. We observe that the binding interactions of the dimer and monomer to the CXCR2 N-terminal domain, which plays an important role in determining affinity and activity, are essentially conserved. The potent activity of the CXCL1 dimer is novel: dimers of the CC chemokines CCL2 and CCL4 are inactive, and the dimer of the CXC chemokine CXCL8 (which is closely related to CXCL1) is marginally active for CXCR1 but shows variable activity for CXCR2. We conclude that large differences in dimer activity among different chemokine-receptor pairs have evolved for fine-tuned leukocyte function.

Chemokine CXCL1 mediated neutrophil recruitment: Role of glycosaminoglycan interactions

The chemokine CXCL1/MGSA plays a pivotal role in the host immune response by recruiting and activating neutrophils for microbial killing at the tissue site. CXCL1 exists reversibly as monomers and dimers, and mediates its function by binding glycosaminoglycans (GAG) and CXCR2 receptor. We recently showed that both monomers and dimers are potent CXCR2 agonists, the dimer is the high-affinity GAG ligand, lysine and arginine residues located in two non-overlapping domains mediate GAG interactions, and there is extensive overlap between GAG and receptor-binding domains. To understand how these structural properties influence in vivo function, we characterized peritoneal neutrophil recruitment of a trapped monomer and trapped dimer and a panel of WT lysine/arginine to alanine mutants. Monomers and dimers were active, but WT was more active indicating synergistic interactions promote recruitment. Mutants from both domains showed reduced GAG heparin binding affinities and reduced neutrophil recruitment, providing compelling evidence that both GAG-binding domains mediate in vivo trafficking. Further, mutant of a residue that is involved in both GAG binding and receptor signaling showed the highest reduction in recruitment. We conclude that GAG interactions and receptor activity of CXCL1 monomers and dimers are fine-tuned to regulate neutrophil trafficking for successful resolution of tissue injury.

Secretion of the Human Toll-like Receptor 3 Ectodomain Is Affected by Single Nucleotide Polymorphisms and Regulated by Unc93b1

The innate immune receptor Toll-like receptor 3 (TLR3) can be present on the surface of the plasma membranes of cells and in endolysosomes. The Unc93b1 protein has been reported to facilitate localization of TLR7 and 9 and is required for TLR3, -7, and -9 signaling. We demonstrate that siRNA knockdown of Unc93b1 reduced the abundance of TLR3 on the cell surface without altering total TLR3 accumulation. In addition, siRNA to Unc93b1 reduced the secretion of the TLR3 ectodomain (T3ECD) into the cell medium. Furthermore, two human single nucleotide polymorphisms that affected herpesvirus and influenza virus encephalopathy as well as a natural isoform generated by alternative splicing were found to be impaired for T3ECD secretion and decreased the abundance of TLR3 on the cell surface. The locations of the SNP P554S and the deletion in the isoform led to the identification of a loop in the TLR3 ectodomain that is required for secretion and a second whose presence decreased secretion. Finally, a truncated protein containing the N-terminal 10 leucine-rich repeats of T3ECD was sufficient for secretion in an Unc93b1-dependent manner.

Dynamic conformational switching in the chemokine ligand is essential for G-protein-coupled receptor activation

Chemokines mediate diverse functions from organogenesis to mobilizing leucocytes, and are unusual agonists for class-A GPCRs (G-protein-coupled receptors) because of their large size and multi-domain structure. The current model for receptor activation, which involves interactions between chemokine N-loop and receptor N-terminal residues (Site-I) and between chemokine N-terminal and receptor extracellular loop/transmembrane residues (Site-II), fails to describe differences in ligand/receptor selectivity and the activation of multiple signalling pathways. In the present study, we show in neutrophil-activating chemokine CXCL8 that the highly conserved GP (glycine-proline) motif located distal to both N-terminal and N-loop residues couples Site-I and Site-II interactions. GP mutants showed large differences from native-like to complete loss of function that could not be correlated with the specific mutation, receptor affinity or subtype, or a specific signalling pathway. NMR studies indicated that the GP motif does not influence Site-I interactions, but molecular dynamics simulations suggested that this motif dictates substates of the CXCL8 conformational ensemble. We conclude that the GP motif enables diverse receptor functions by controlling cross-talk between Site-I and Site-II, and further propose that the repertoire of chemokine functions is best described by a conformational ensemble model in which a network of long-range coupled indirect interactions mediate receptor activity.

Chemokine CXCL7 Heterodimers: Structural Insights, CXCR2 Receptor Function, and Glycosaminoglycan Interactions

Chemokines mediate diverse fundamental biological processes, including combating infection. Multiple chemokines are expressed at the site of infection; thus chemokine synergy by heterodimer formation may play a role in determining function. Chemokine function involves interactions with G-protein-coupled receptors and sulfated glycosaminoglycans (GAG). However, very little is known regarding heterodimer structural features and receptor and GAG interactions. Solution nuclear magnetic resonance (NMR) and molecular dynamics characterization of platelet-derived chemokine CXCL7 heterodimerization with chemokines CXCL1, CXCL4, and CXCL8 indicated that packing interactions promote CXCL7-CXCL1 and CXCL7-CXCL4 heterodimers, and electrostatic repulsive interactions disfavor the CXCL7-CXCL8 heterodimer. As characterizing the native heterodimer is challenging due to interference from monomers and homodimers, we engineered a “trapped” disulfide-linked CXCL7-CXCL1 heterodimer. NMR and modeling studies indicated that GAG heparin binding to the heterodimer is distinctly different from the CXCL7 monomer and that the GAG-bound heterodimer is unlikely to bind the receptor. Interestingly, the trapped heterodimer was highly active in a Ca2+ release assay. These data collectively suggest that GAG interactions play a prominent role in determining heterodimer function in vivo. Further, this study provides proof-of-concept that the disulfide trapping strategy can serve as a valuable tool for characterizing the structural and functional features of a chemokine heterodimer.

Platelet-Derived Chemokine CXCL7 Dimer Preferentially Exists in the Glycosaminoglycan-Bound Form: Implications for Neutrophil–Platelet Crosstalk

Platelet-derived chemokine CXCL7 (also known as NAP-2) plays a crucial role in orchestrating neutrophil recruitment in response to vascular injury. CXCL7 exerts its function by activating the CXC chemokine receptor 2 (CXCR2) receptor and binding sulfated glycosaminoglycans (GAGs) that regulate receptor activity. CXCL7 exists as monomers, dimers, and tetramers, and previous studies have shown that the monomer dominates at lower and the tetramer at higher concentrations. These observations then raise the question: what, if any, is the role of the dimer? In this study, we make a compelling observation that the dimer is actually the favored form in the GAG-bound state. Further, we successfully characterized the structural basis of dimer binding to GAG heparin using solution nuclear magnetic resonance (NMR) spectroscopy. The chemical shift assignments were obtained by exploiting heparin binding-induced NMR spectral changes in the WT monomer and dimer and also using a disulfide-linked obligate dimer. We observe that the receptor interactions of the dimer are similar to the monomer and that heparin-bound dimer is occluded from receptor interactions. Cellular assays also show that the heparin-bound CXCL7 is impaired for CXCR2 activity. We conclude that the dimer–GAG interactions play an important role in neutrophil–platelet crosstalk, and that these interactions regulate gradient formation and the availability of the free monomer for CXCR2 activation and intrathrombus neutrophil migration to the injury site.

Glycosaminoglycan Interactions Fine-Tune Chemokine-Mediated Neutrophil Trafficking: Structural Insights and Molecular Mechanisms

Circulating neutrophils, rapidly recruited in response to microbial infection, form the first line in host defense. Humans express ~50 chemokines, of which a subset of seven chemokines, characterized by the conserved “Glu-Leu-Arg” motif, mediate neutrophil recruitment. Neutrophil-activating chemokines (NACs) share similar structures, exist as monomers and dimers, activate the CXCR2 receptor on neutrophils, and interact with tissue glycosaminoglycans (GAGs). Considering cellular assays have shown that NACs have similar CXCR2 activity, the question has been and remains, why do humans express so many NACs? In this review, we make the case that NACs are not redundant and that distinct GAG interactions determine chemokine-specific in vivo functions. Structural studies have shown that the GAG-binding interactions of NACs are distinctly different, and that conserved and specific residues in the context of structure determine geometries that could not have been predicted from sequences alone. Animal studies indicate recruitment profiles of monomers and dimers are distinctly different, monomer–dimer equilibrium regulates recruitment, and that recruitment profiles vary between chemokines and between tissues, providing evidence that GAG interactions orchestrate neutrophil recruitment. We propose in vivo GAG interactions impact several chemokine properties including gradients and lifetime, and that these interactions fine-tune and define the functional response of each chemokine that can vary between different cell and tissue types for successful resolution of inflammation.

Lysines and Arginines play non-redundant roles in mediating chemokine-glycosaminoglycan interactions

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.

Heparin-bound chemokine CXCL8 monomer and dimer are impaired for CXCR1 and CXCR2 activation: Implications for gradients and neutrophil trafficking

Chemokine CXCL8 plays a pivotal role in host immune response by recruiting neutrophils to the infection site. CXCL8 exists as monomers and dimers, and mediates recruitment by interacting with glycosaminoglycans (GAGs) and activating CXCR1 and CXCR2 receptors. How CXCL8 monomer and dimer interactions with both receptors and GAGs mediate trafficking is poorly understood. In particular, both haptotactic (mediated by GAG-bound chemokine) and chemotactic (mediated by soluble chemokine) gradients have been implicated, and whether it is the free or the GAG-bound CXCL8 monomer and/or dimer that activates the receptor remains unknown. Using solution NMR spectroscopy, we have now characterized the binding of heparin-bound CXCL8 monomer and dimer to CXCR1 and CXCR2 receptor N-domains. Our data provide compelling evidence that heparin-bound monomers and dimers are unable to bind either of the receptors. Cellular assays also indicate that heparin-bound CXCL8 is impaired for receptor activity. Considering dimer binds GAGs with higher affinity, dimers will exist predominantly in the GAG-bound form and the monomer in the free form. We conclude that GAG interactions determine the levels of free CXCL8, and that it is the free, and not GAG-bound, CXCL8 that activates the receptors and mediates recruitment of blood neutrophils to the infected tissue.