DELINEATING THE MOLECULAR INTERACTIONS OF GRO CHEMOKINES

Abstract

Chemokines are small 8-10 kD signaling entities that are involved in numerous physiological and pathological processes including leukocyte trafficking, angiogenesis, organogenesis, tissue development, and tumorigenesis etc, through their association with cell surface glycosaminoglycans (GAGs) and the G-protein coupled receptors (GPCRs). Humans express around 50 chemokines which are segregated into different classes namely CXC, CC, CX3C, and C chemokines based on the positioning of N-terminus cysteine residues. Chemokine monomers share the canonical tertiary structure comprising of long unstructured N-terminus, followed by 310-helix, three antiparallel -sheets, and a C-terminal -helix. Chemokines oligomerize into dimers, tetramers, and other higher order oligomers. They form two types of dimers, namely CC and CXC. CXC dimer is more globular and is formed by the two antiparallel -helices lying on the top of six stranded antiparallel -sheets, whereas CC dimers involve the formation of a two stranded antiparallel -sheet between the N-terminal regions of the two monomeric units. Recent studies have also reported the formation of heterodimers between several CC/CXC chemokines thus adding complexity to the underlying process of chemokine regulated leukocyte trafficking processes. Neutrophil activating chemokines (NACs) belong to the subfamily of CXC chemokines which are specifically involved in recruitment of neutrophils. NAC family comprises of seven proteins namely CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7 and CXCL8, with a signature ELR (Glutamic acid- Leucine- Arginine) motif at their N-terminus. Among NACs, CXCL1 (GRO , CXCL2 (GRO , and CXCL3 (GRO ) are called as Growth Related Oncogene chemokines (GRO). GRO family arose as a result of two rounds of gene duplication during the course of evolution. GRO chemokines are closely related to each other, and are involved in growth and progression of melanoma tumors. Despite their super-close relativity in sequence and structure, biological studies have reported the differential expression patterns of all three GRO genes in tissue and signal specific manner, and their involvement in different functions. They have also adapted different mechanisms and signaling pathways to accomplish their specific functions. The differential behaviors of these highly related members of GRO chemokines can be implicated to their differential evolution patterns, oligomerization (homo/hetero), and variable interactions with cellular partners (GAGs and receptors). Although plethora of literature is available about the differential function of GRO proteins, however, the mechanistic details of their evolution and viii structure-function relationships at molecular level are scarce. Hence, the research work in the current thesis has been designed to decipher the evolutionary perspectives, oligomerization potencies, differential GAG binding interactions, and structure-stability relationships of GRO chemokines at atomic level by using multitude of biophysical and computational techniques. Specific details of thesis chapters (1 to 4) are as follows. Chapter 1 provides an overview of chemokine research area including the role of chemokines in innate and adaptive immunity, origin of chemokine family, classification of chemokines, their structural and other biological properties including oligomerization, interactions with chemokine receptors and GAGs. A detailed literature survey of different types of NACs, their biological importance, structures solved by X-ray crystallography and NMR spectroscopy, studies focused on interaction of NACs with cellular binding partners including receptor and GAGs have also been discussed with special emphasis on known literature of GRO chemokines. This chapter also provides the snapshot of various bioinformatics and biophysical tools used in the thesis to accomplish the proposed objectives. Chapter 2 presents the evolution-structure relationships of GRO chemokines. The evolutionary mechanisms including selection pressures involved in diversification of GRO genes have been characterized by performing comprehensive evolutionary analysis among different mammalian species. Phylogenetic analysis showed species specific evolution pattern in GRO chemokines, while the selection analysis revealed that these genes have undergone concerted evolution and possess positive selection sites, although majority of them are under purifying selection. Interestingly, positively selected sites are more concentrated on the C-terminal / GAG binding and dimerization segment. Substitution rate analysis also corroborated the species specific evolution, and confirmed C-terminal domain of GRO genes as the highest substituted segment. Further, the structural analysis established that these nucleotide alterations in the GAG binding domain are the source of surface charge modulation, thus generating the differential GAG binding surfaces and multiple binding sites as per evolutionary pressure, even though the helical surface is primordial for GAG binding. Indeed, such variable electrostatic surfaces are crucial to regulate chemokine gradient formation during host’s defense against pathogens thus explaining the significance of chemokine promiscuity. Chapter 3 characterizes the homo / hetero oligomerization potencies and GAG binding features of CXCL1 and CXCL2. Genes for murine CXCL1 and CXCL2 were successfully cloned, expressed, and proteins were purified using the combination of various chromatography ix techniques. Homo-oligomerization propensity of CXCL1 and CXCL2 assessed by size exclusion chromatography (SEC) and glutaraldehyde cross linking assay demonstrated the strong dimeric nature of CXCL2 as compared to CXCL1. NMR 1H-15N HSQC experiment of CXCL1 and CXCL2 at same concentration (~ 150 μM) evidenced two set of peaks corresponding to both monomer and dimer for CXCL1, whereas a single set of dimeric resonances in CXCL2 spectrum establishing their intrinsic differential homo dimerization capabilities. To further characterize the resonances from the monomeric and/or dimeric species and dimer interface residues, NH cross peaks in the 1H-15N HSQC spectra of both CXCL1 and CXCL2 were assigned. The formation of CXCL1/CXCL2 heterodimer was characterized experimentally by mixing their equimolar concentrations and monitoring the 1H-15N resonances of CXCL1. The addition of unlabeled CXCL2 resulted in new set of dimer-interface peaks along with the existing two sets of resonances (CXCL1 monomer/homo-dimer), accompanied by a significant attenuation in the peak intensities. Quantification of intensities of the dimer interface residues Q25 and L30 of CXCL1 before and after the addition of CXCL2 indicated the presence of ~ 40 % heterodimer under chosen experimental conditions, thus directly demonstrating the potential formation of CXCL1-CXCL2 heterodimers. Upon assessing the oligomerization characteristics of CXCL1/2 homo/hetero dimers, different oligomeric species of these chemokines were subjected to interactions with GAGs/ GAG mimetics (HP6-heparin hexasaccharide, HA6-hyaluronan hexasaccharide, SHA6-synthetic sulfated hyaluronan hexasaccharide, and NC6-neocarradodecaose hexasulfate) in order to monitor the phenomenon of GAG induced oligomerization. NMR titration experiments indicated that HP6 and SHA6 induced dimerization in CXCL1, CXCL2, CXCL1/2 heterodimer in contrast to HA6 and NC6. These results indicated that sulfation of GAGs indeed is essential to oligomerize/dimerize chemokines. Moreover, the results evidenced that the extent and positioning of the sulfation also play a crucial role in regulating the chemokine GAG interactions and GAG induced chemokine oligomerization. Chapter 4 depicts the biophysical characterization of CXCL3 and its comparative analysis of the structure-stability-dynamics relationship with CXCL2. Murine CXCL3 gene was successfully cloned, expressed, and purified partly as soluble form (supernatant) from cytoplasm and rest as insoluble form from inclusion bodies to maximize the protein yield. Resonance assignments of CXCL3 NH cross peaks were obtained using conventional 3D-NMR experiments. Oligomerization state of CXCL3 appraised using size exclusion chromatography, 2D-DOSY, and x 15N-NOESY indicated that the dimeric nature of CXCL3 is similar to CXCL2. Heparin binding assay suggested the GAG binding affinity of CXCL3 and CXCL2 are weaker compared to CXCL1. Temperature dependent Circular Dichroism (CD) studies evidenced that both the CXCL2 and CXCL3 structures are highly stable to thermal perturbations. Structural alignment and contact map analysis of these two chemokines implied their structural equivalence with differential electrostatic surface potentials on both -sheet and -helix surfaces. Further, tertiary structural characteristics of CXCL3 and CXCL2 investigated using ANS fluorescence showed distinct fluorescence profiles. Binding mode of ANS to CXCL3 and CXCL2 explored using fluorescence life time analysis (FLS) and NMR suggested the presence of surface exposed specific hydrophobic binding pocket on helical surface of CXCL3 in contrast to CXCL2. To further gain insights into their residue-level differential stability-dynamics properties of CXCL2 and CXCL3, NMR based native state hydrogen exchange (NHX) studies, temperature dependence NMR and 15N backbone relaxation studies were performed. Stabilization free energies evidenced that CXCL2 is comparatively more stable than CXCL3. Temperature dependence, amide proton chemical shifts, and 15N relaxation studies unraveled the differential dynamic features of these two paralogs at both faster (ps-ns) and slower (s-ms) time scales. These differential structural, stability and dynamic features demonstrate that although CXCL2 and CXCL3 share similar tertiary structural and oligomerization features, they do possess specific electrostatic surfaces, varied dynamics and stability characteristics that can be implicated to their differential / specific functional behaviors. In summary, the present thesis unraveled the evolutionary, structural, and functional relationship of GRO chemokines and characterized their structural, dynamic, oligomerization, and GAG binding features at residue level, thus providing mechanistic insights into their molecular interactions. This comprehensive study will aid chemokine/GAG researchers in rational designing of novel chemokine and/or GAG mimetics to regulate neutrophil trafficking.