EVOLUTION-STRUCTURE-STABILITY-FUNCTION PARADIGM OF CXC CHEMOKINE INTERLEUKIN-8

Abstract

Chemokine system is a vast and essential part of the defense system in any multicellular organism and consists of mainly chemokine ligands and their receptors. Chemokines are the group of low molecular weight chemotactic cytokines, mainly involved in the recruitment of leukocytes to the site of inflammation / infection, and also play important roles in angiogenesis and maintaining the homeostasis. Chemokines-directed migration of leukocytes significantly helps in progression of inflammatory or immune related diseases, including atherosclerosis, cancer, autoimmunity, and many others. They are also known as secondary pro-inflammatory cytokines as their productions generally induced by the primary inflammatory cytokines such as IL-1 and TNF-α. The chemokines as well as their receptors are continuously evolving molecules, forming a substantial and complex repertoire in the genome with more than 50 chemokines in the human genome. The root of chemokine evolution lies in the early ancestor of vertebrates that evolved around 650 mya. They are one of the important genes that evolved with the advent of the adaptive immune system in vertebrates. Chemokines are divided into four groups; CC, CXC, CX3C, and XC on the basis of cysteine residue positioning at the N-terminal. Among these, the CXC chemokine group has been divided into two subgroups, ELR(+)CXC and ELR(-)CXC chemokines depending upon the presence of ELR motif, preceding the first cysteine residue at the N-terminal. ELR(+)CXC chemokines can activate and recruit neutrophils, whereas ELR(-)CXC members can only induce the migration of lymphocytes but cannot activate neutrophil cells in mammals. The oligomerization is another inherent and crucial characteristic of chemokines plays important roles in the regulation of cell signaling and chemotactic cell migration. All the neutrophil-activating ELR(+)CXC chemokines form CXC-type dimers or higher order oligomers at higher concentrations in vitro and in vivo. Oligomerization (monomers and dimer equilibrium) modulates the affinity of chemokine molecule interactions toward various glycosaminoglycans (GAGs), and GPCR receptor activation. Interleukin-8 (IL8/CXCL8) is the most prominent member of “ELR(+)CXC chemokine/neutrophil activating chemokine (NAC), which regulate neutrophil trafficking during infections/inflammation by binding to cell surface GAGs and G-protein coupled receptors CXCR1/CXCR2. IL8 plays an important role in wound healing, angiogenesis, neovascularization, metastasis and inflammation. NMR/X-ray structure of human IL8 protein consists of mainly four parts (a) long unstructured N-terminal, (b) 310 helix, (c) three antiparallel β-sheets and (c) C-terminal α-helix. The N-terminal loop along with residues from 30s and 40s loop are involved in receptor interaction, while positively charged residues from 310-helix and C-terminal helix are majorly responsible for GAG binding. Although the ELR motif is a conserved feature of avian and mammalian ELR(+)CXC chemokines (IL8 protein), this motif is majorly absent in teleost fish except for Haddock (Melanogrammus aeglefinus), and Atlantic cod (Gadus morhua). The first non-mammalian IL8 sequence was discovered in the lamprey in 1999, since then a large number of IL8 sequences are reported in the teleost fish, including some novel CXC chemokines whose structural and functional homologs are absent in humans. It has been reported that some teleost IL8 have substitution form of ELR motif such as DLR/NLH and revealed that they are able to activate the neutrophils. A large number of domestic animals such as dog, cat, horse, bovine, and other animals from the laurasiatherian superorder also possess orthologs of the human IL8 genes. It is involved in the progression of disease and cancers thus acting as an important prognostic biomarker for these various cancers. The various studies have reported that the IL8 genes in all species are derived from a single ancestor through speciation and further evolved via random mutations, suggesting that the core functions of IL8 are conserved in all species. However, there are phenotypic variations exist within the orthologs that can be classified as fluctuations in function, structure, stability, and binding etc. Although, great deal of literature is reported on the structure, stability, and function of human IL8 protein, only fragmentary information is available from the primitive, laurasian, and other group of animals. The present study is dedicated to understand the evolutionary origin of ELR(+)CXC chemokine (IL8) and the structure-stability-function paradigm of IL8 orthologs. It includes the evolutionary and functional divergence analysis of IL8 gene in lower vertebrates (fish) through sequence and positive selection methods. The comparative structural-stability-function studies between IL8 orthologs were performed using multidimensional solution state NMR techniques. Further,specific molecular interaction of IL8 with flavonoid baicalin has also been elucidated. The specific details of the chapters (Chapter 1 - 5) are described as follows. Chapter 1 provides an overview of evolution of the immune system in lower animals as well as in higher vertebrates and their important components (cells and molecules) along with their molecular interactions. Existing literature on molecular evolution of chemokines, chemokine receptors, chemokine classifications, and their important biological functions has been discussed in great details. An outline of ELR+CXC chemokine and their roles in neutrophil trafficking to the site of infection/inflammation by interacting with the glycosaminoglycans (GAGs) and G-protein coupled receptors (GPCRs) is provided. The structural basis and importance of oligomerization in stability and functions of chemokines was also discussed. Further details about structural and functional features, clinical significance of IL8, structural and biological importance of various types of trapped / engineered IL8 monomers have been provided. The interactions of chemokines with small potential therapeutic compounds were illuminated. Chapter 2 of the thesis is devoted to unravelling the evolutionary origin of ELR motif using CXC chemokine CXCL8. The “ELR” motif of NAC chemokines in mammals is essential for the CXCR1/CXCR2 receptor activation. 76 full-length gene sequences of CXCL8 sequences from primitive species (fish) were collected from various databases and analyzed using multiple sequence alignment analysis (MSA). In order to understand the evolutionary origin of “ELR” motif in the CXC chemokines, a thorough evolutionary study of CXCL8 gene from the various fishes and primates was performed. Phylogenetic analysis revealed that the CXCL8 gene can be classified into four distinct lineages (CXCL8-L1a, CXCL8-L1b, CXCL8-L2, and CXCL8-L3), where CXCL8-L1a is the fastest evolving lineage and CXCL8-L3 is the slowest. Positive selection analysis suggested that the “ELR/DLR” motif containing branches (gadoid and coelacanth) are positively selected. The probable evolutionary trend of “ELR” motif suggested that this motif in ancestor CXCL8 is evolved from the GGR of Lamprey (Agnatha), followed by duplication giving rise to two main motifs in CXCL8 “NXH” in L3 lineage and “ELR/DLR” in L1a/L1b lineages. Although, structural analysis suggested that the overall topology of the CXCL8 proteins is similar, differences do exist at the individual structural elements among the members of different lineages. Functional distance analysis suggested that the CXCL8-L3 lineage is more distant compared to the CXCL8-L1a and L1b lineages from the inferred ancestor. Functional divergence analysis between different lineages suggested that most of the selected residues are important for receptor or glycosaminoglycan binding. Such a functional diversification can be attributed to the novel set of functions adopted by CXCL8 in various species. In summary, the results evidenced that the branch containing the ELR motif was a result of positive branch selection, and several of the residues exhibiting functional divergence are underlying in the receptor or glycosaminoglycan binding domains of CXCL8. Chapter 3 describes the molecular insights into the differential structure-dynamics-stability-function features of IL8 orthologs. IL8 exhibits significant functional divergence among different orthologs and paralog proteins. The comparative molecular analysis was performed on canine (laurasians) and human (primates) IL8 proteins using molecular evolutionary analysis and solution NMR spectroscopy methods. The in-silico functional divergence analysis suggested that the functional divergence can be observed between the different groups of organisms and order follows, Primates to Laurasians > Primates to Fish_ L1b > Laurasians to Fish_ L1b. The recombinant proteins of IL8 from canine (cIL8) and human (hIL8) were cloned and expressed, and then characterized using NMR spectroscopy. The residue level NMR studies suggested that, although the overall structural architecture is similar in both the orthologs, several of the essential hydrophobic and electrostatic interactions are altered due to amino acid substitutions. The differential contacts resulted in systematic differences in backbone dynamics and low-energy excited states accessed by the IL8 variants. Further, the modulation of these amino acid substitutions resulted in decrease of stability and heparin binding affinity in the canine IL8 compared to its human counterpart. Indeed, structural and sequence analysis evidenced for specificity of molecular interactions for their binding with cognate receptor (CXCR1) and glycosaminoglycan (heparin), thus suggesting for a noticeable functional specificity and divergence between the two IL8 orthologs. Chapter 4 portrays the comparative structure-stability-dynamic study of human IL8 WT (dimer) and a reported engineered trapped monomer. The monomeric IL8 molecule was produced by substitution of dimer interface residues Leu30 and Val32 with Tyr30 and arg32 respectively. Molecular characterization studies suggested that the mutated IL8 protein clearly exists as monomer that is supported by the loss of NOE contacts between residues at dimer interface. Although the overall structural topology of monomer and dimer is similar but the secondary structure analysis suggested that the C-terminal α-helix is shorter in IL8 monomer (by 5 residues) in comparison to its dimer. Het-NOE exhibited noticeable differences only at N and C-terminal. It is found that the more number of residues share alternative conformations in IL8 monomer compared to IL8 dimer suggesting the enhanced native state ruggedness of the monomer. Further, native state hydrogen exchange analysis suggested that most of the residues of IL8 monomer are easily accessible to the solvent and exchangeable, suggesting that the monomer is comparatively less stable than the dimer. Furthermore, heparin binding assay suggested that affinity of monomer towards heparin is lower to that of the dimer. Chapter 5 elucidates the molecular interaction analysis between flavonoid (baicalin) and IL8 (monomer/dimer). Baicalin is widely used as traditional medicine in Asian countries for treatment of various inflammatory diseases, cardiovascular, and neurodegenerative disorders, extracted from the roots of Scutellaria baicalensis. It is believed that it exhibit anti-inflammatory activity by binding to chemokines via disrupting the chemokine-receptor interactions. However, the exact mechanism and residue/atomic level interactions have been not characterized yet. In order to understand the binding interactions between IL8 and baicalin, tryptophan fluorescence quenching, HSQC based chemical shift perturbation map (CSP), and molecular docking studies were performed with. The fluorescence quenching experiments suggested that baicalin binding to IL8 follows the static quenching mechanism with quenching constant of 5.8x1013 for monomer and 5.9x1013 for dimer and the number of binding site is ~ 1 per monomer. The dissociation constants of IL8 interactions are; 5 ± 1 μM for monomer and 4 ± 1 μM for dimer suggesting that both the oligomeric forms binds with similar affinities. The CSP map revealed that the binding of baicalin to the IL8 is specific, and mainly interacts with the residues from N-terminal, 40s loop, 50s loop, and β3 strand. Furthermore, molecular docking results are also in the line with the CSP results. It is observed that the hydrogen and hydrophobic interactions dominates baicalin-IL8 binding with same residues involved in the receptor interaction. In order understand the effect of heparin (GAGs) in binding of baicalin to IL8; the binding experiments were performed in presence of heparin sodium salt. The results suggested that heparin is not interfering in baicalin-IL8 binding, as no significant change has been noticed in the binding parameters. Thus, our findings suggest that the baicalin mainly interacts with IL8 by using the receptor binding pocket, and can interfere with the IL8-receptor interactions. In summary, current research work delineates the evolutionary origin of “ELR” motif in CXC chemokine by using CXCL8 gene/protein sequences from the lower vertebrates and further classified CXCL8 gene in four lineages instead of previously described only three lineages. IL8 orthologs from two diverged mammalian species (canine and human) have considerable difference in structure-stability-dynamics features and also exhibit species-specific receptor recognition and GAG binding affinity. IL8 monomer possesses shorter C-terminal α-helix and comparatively less stable than the dimer. Flavonoid Baicalin binds to monomeric and dimeric IL8 with similar affinities.