DISSECTING THE MOLECULAR INTERACTIONS OF CCL2 CHEMOKINE WITH FLAVONOIDS

Abstract

The traditional diorama of the innate immune system during epidemiological conditions and inflammation comprises a diligent activation and recruitment of various immune cells to the foci of inflammation. The process of elimination of invaded pathogens, injurious conditions and other pathophysiological insults is carried out through the directed recruitment of leukocytes to the inflamed site, which is commonly known as leukocyte trafficking. Chemokines, widely known as chemotactic cytokines, are the crucial players in the leucocyte migration process that regulate and mediate the migratory patterns and positioning of leucocytes. The decisive functions of chemokines include an array of undulatory cycles ranging from the positioning of immune cells in various disease conditions to the development and homeostasis of the immune system. Chemokines are known as the largest family of cytokines, comprising of more than 50 members in human and murine. These are the small, extremely conserved and soluble chemotactic proteins with a molecular weight ranging from 8 to 10 kDa. These chemotactic proteins are extensively associated with several deleterious pathological conditions. According to the positioning of the first two of four cysteine residues, they are categorized into four sub-families termed as; C, CC, CXC and CX3C chemokine. In order to mediate their biological activities, chemokines are known to interact with their cognate receptors and glycosaminoglycans (GAGs). Chemokine receptors belong to the γ subfamily of G-protein coupled with the seven-transmembrane domain and are known as serpentine receptors. To date, ~ 50 chemokines, ~ 20 chemokine receptors involved in signaling pathways have been identified in humans, thus establishing the promiscuous chemokine-receptor relationship. CC chemokines are also recognized as β-chemokines; they comprise of two consecutive cysteine amino acids at N-terminal, and two disulfide bridge forms between 1st and 3rd cysteine and 2nd and 4th cysteine residues respectively. For CC chemokines, ~ 27 different ligands/members have been reported in mammals (CCL1 to CCL28). In general, most of the members of the CC chemokine family comprise of the four cysteine residues and known as C4-CC chemokine; however, some members exhibit six cysteines and termed as C6-CC chemokines. The basic structure of CC chemokines consists of a long N-terminal, followed by three antiparallel -sheets and C-terminal -helix. These chemotactic proteins primarily induce the activation and migration of monocytes/macrophages. However, they can also stimulate the migration of natural killer (NK) cells and dendritic cells. The chromosomal location of CC chemokines genes has been mapped on human chromosome 17q11.2. Further, on the basis of the location on chromosome, two gene groups have been identified; they include - monocyte chemotactic proteins (MCPs), and macrophage inflammatory proteins (MIPs). MCP sub-family of CC chemokines comprises a group of four members named MCP-1/CCL2, MCP-2/CCL8, MCP-3/CCL7 and MCP-4/CCL7. Among these four members, MCP-1 widely recognized as CCL2 is a first discovered human CC chemokine and is the most crucial member of the MCP family. It is a 76 amino acid long protein with ~9 kDa molecular weight. CCL2 is expressed and produced by various types of cells such as smooth muscle cells, fibroblast, endothelial and mesangial cells; however, the macrophages/monocytes are recognized as its prime source of production. CCL2 has been associated with many deleterious ailments such as cardiovascular, atherosclerosis, multiple sclerosis, tumor/cancerous conditions, and HIV infections. To intercede these pernicious conditions, CCL2 interact significantly with its cognate receptor CCR2 and GAGs, and explicitly controls the migration of monocytes to the site of infection. The canonical structure of CCL2 comprises of a long and disordered N-terminal, consecutive four -sheets followed by a C-terminal -helix. Since oligomerization has accepted as the most crucial process in the regulation of various cell signaling pathways. Human CCL2 chemokine has been reported to exist as homodimer above 100 μM concentration. Further, it has been reported that in its monomeric conformation, CCL2 explicitly interact with CCR2 and arbitrates various biological functions. In contrast to human, the murine CCL2, which is a close ortholog of human CCL2, is 125 amino acid long and comprises of 49 extra residues at C-terminal end than that of human CCL2. The murine CCL2 is commonly called as a JE gene and exhibits high sequence similarity to that of its human partner. Though the significance of the existence of this extended C-terminal in murine is not clear, however, it has been reported that it is highly decorated with O-linked glycosylation. Flavonoids are the group of naturally occurring plant products and are the crucial secondary metabolites of plants. Owing to their tremendous biological activities such as anti-oxidant, anti-inflammatory, anti-cancer and antimicrobial effects, they are the prominent candidates of alternative medicine. These natural compounds also recognized as the main ingredients of the human diet. The interaction of flavonoids with biological macromolecules such as proteins, DNA, RNA and membrane lipids has provided new insight into the mode of the interaction of these binding partners and instilled new therapeutic formulations in the form of nutraceutical development. Although a great deal of literature has been reported on the molecular interaction of flavonoids with many proteins, no such information is available for their molecular interaction with immunomodulatory proteins such as chemokines. The present thesis unravels of the comparative evolutionary aspects of CCL2 chemokines, and their glycosylation patterns of C-terminal region in the rodentia families. The study includes the evolutionary and functional divergence analysis of the CCL2 gene in primates and rodentia families. Both the human and murine CCL2 genes were cloned; proteins were purified in both wild type and monomeric form (P8A mutant). The differential biophysical features of both orthologs (human and murine) in their dimeric and monomeric conformation were investigated using the combination of biophysical approaches such as circular dichroism spectroscopy, fluorescence spectroscopy and NMR spectroscopy in association with bioinformatics approaches. These CCL2 proteins were assessed for their potency to directly bind a known immuno-modulatory flavonoid baicalin, which is previously reported to regulate the chemokine-receptor interactions through cell based assays. Further, the molecular interaction of two subgroups namely flavones and flavonols with CCL2 orthologs has been delineated. The details of these studies are arranged in the form of the chapter (Chapter 1-4) in the thesis, and are described as follows. Chapter 1 describes the details of the immune system and its components, namely the innate immune system and the adaptive immune system. The prevailing literature on the chemokines and their classification, structural details, chemokine receptors, and the relevance of chemokine-receptor interactions to arbitrate the biological functions has been explained in detail. Further, the detailed information about the overview of CC chemokines, types of CC chemokines and their functions has been described. An outline of the structural features of CCL2 and the detail of the types of post-translational modifications in CCL2 chemokine has also been extensively reviewed. Likewise, the role of CCL2 in monocyte recruitment by interacting with the glycosaminoglycans (GAGs) and its cognate receptor was discussed in detail. The role of CCL2 in various pernicious diseases and its clinical significance has also been elucidated. The last section of Chapter 1 describes the detailed overview of the types of flavonoids, their chemical structure and their significant biological functions. Chapter 2 is devoted explicitly for delineating the differential evolutionary facets of the CCL2 chemokine gene from primates and rodentia family. Since no structural details are available for the rodentia family CCL2 proteins, Chapter 2 has been designed to unravel the structural, dynamics and in-silico functional characteristics of murine CCL2 chemokine. This chapter also provides comparative molecular insights into the differential dynamics features of CCL2 orthologs. The study has been investigated by using a comprehensive set of molecular biology, biophysical and bioinformatics approaches. It includes evolutionary and functional divergence and the NMR spectroscopy techniques approaches. The evolutionary approaches have revealed that the primary sequences of CCL2 orthologs among rodents and primates vary significantly at the C-terminal region. Also, the disordered analysis and glycosylation studies have unveiled that the C-terminal of the rodentia family CCL2 proteins is highly disordered and extensively decorated with O-linked glycosylation. Further, the molecular biology and biophysical investigations have unraveled that the N-terminal portion of the murine CCL2 forms a canonical CC chemokine dimer similar to that of human CCL2. However, unlike human CCL2, the murine ortholog exhibits extensive dynamics in the μs-ms timescales. The last section of Chapter 2 has described comparative and detailed information about the differences in the receptor-binding and glycosaminoglycan binding surfaces of these orthologs proteins. In conclusion, Chapter 2 has provided the role of molecular evolution in shaping orthologous proteins with differential structural and dynamics characteristics to engage them in specific molecular interactions. Chapter 3 is dedicated to elucidate the binding surface for baicalin flavonoid in CCL2 ortholog proteins (human and murine). Since Chapter 2 has revealed that CCL2 protein exhibits the monomer-dimer equilibrium, the interaction of baicalin was investigated using both CCL2 dimers and monomers. To accomplished that, the monomeric variant of murine CCL2 (mCCL2) has been constructed using previously reported obligate monomeric variants of human CCL2 (hCCL2) by generating P8A mutants on both orthologs. Our comparative biophysical studies on mCCL2 and hCCL2 proteins have evidenced that the constructed variants exclusively existed in monomeric conformation. Fluorescence spectroscopy multidimensional solution NMR spectroscopy techniques and molecular docking tools were applied to unravel the molecular interactions. The fluorescence results have unveiled that baicalin binds specifically to CCL2 monomers and dimers with similar affinities (Kd ~ 270 ± 20 nM). However, the NMR and molecular docking based results have explicitly deciphered that the baicalin binding surface shows an extensively overlapping with that of the receptor-binding surface on CCL2 chemokine. Thus, Chapter 3 has corroborated the earlier cell-based reports of baicalin interference in chemokine-receptor interactions, and provided a molecular mechanism for the observed attenuation of their chemotactic activity in baicalin presence. Chapter 4 of the thesis has delineated the comparative and comprehensive binding studies of the two subgroups of flavonoids with CCL2 orthologs. As Chapter 3 has unveiled that the baicalin binds to chemokine CCL2 specifically with a strong binding affinity, in this current chapter, some potential flavonoid candidates from two major subgroups of flavonoids known as (a) flavones (baicalein, luteolin and apigenin) and (b) flavonols (myricetin, quercetin and kaempferol) has been screened against CCL2 orthologs (monomers and dimers). To unravel the molecular interactions between flavones and flavonols with CCL2 orthologs, fluorescence spectroscopy, NMR spectroscopy and molecular docking techniques has been applied. The fluorescence quenching experiments have deciphered that the binding affinities drastically vary among the chosen flavones and flavonols. The obtained dissociation constants are in the range of 10-5 to 10-7 M. NMR experiments and molecular docking studies have unveiled the molecular interactions and the binding sites for both subgroups on CCL2 protein. The observed binding surface of these flavonoids to CCL2 orthologs is similar to that of the binding site of baicalin, and also extensively overlapped with that of the receptor-binding surface. Chapter 4 has provided new insights into the binding of flavones and flavonols to CCL2, and also appraised the chemokine researchers to dissect their molecular mechanisms of flavonoid-chemokine interactions in various immunomodulatory processes. In summary, the present research work elucidated the evolutionary facets of CCL2 chemokine from Primate and Rodentia families, and also rationalized the intrinsic advantage of species-specific evolution of chemokine proteins and their functional divergence features. CCL2 orthologs have shown a comparable difference in dynamic features and receptor-binding and GAGs binding surfaces, although the overall structure of both is well conserved.Flavonoids and their glycosides interact with monomeric/dimeric CCL2 orthologs specifically and with significant affinities in the site where their cognate receptors bind. Henceforth, these molecular interactions of flavonoids with chemokines warrant further studies on other subtypes of chemokines along with cell based and animal studies to decipher how they attenuate the cell signaling processes that chemokines induce/regulate.