Comparative structural and functional attributes of Growth-Related Oncogene γ chemokine protein orthologs

Growth-Related Oncogene γ (GRO-γ), commonly known as CXCL3, is a small secreted protein of GRO family involved in migration of neutrophils and oncogenic modulation via their interaction with glycosaminoglycans (GAGs) and cognate CXCR2 receptor. To probe the translation of sequence disparity of the orthologous CXCL3 proteins in terms of structural and biophysical behavior, recombinant human CXCL3 (hCXCL3) was cloned, purified, and compared with murine CXCL3 (mCXCL3). The results suggested that these orthologs were comparable in their structural architecture, dimerization behavior and thermal stability, as evident from AlphaFold 3 predictions, NMR-based diffusion experiments and thermal CD analysis. However, the molecular dynamics simulation analysis established that, mCXCL3 exhibited significantly higher conformational dynamics as compared to hCXCL3, and the C-terminal helix of the hCXCL3 is static and stable compared to mCXCL3 that exhibited tilting motions. Further, both the orthologs differ significantly in their proteolytic stability, unfolding behavior, hydrophobic and electrostatic surface characteristics. Such differential electrostatics resulted in higher GAG binding affinity of hCXCL3 as compared to its murine counterpart. The binding of GAGs to CXCL3 proteins has been established using ITC and NMR experiments. Thermodynamic interpretation of the heparin binding suggested micromolar affinities of the ligand towards both the proteins. Moreover, the two recombinant proteins displayed promising cell migration and proliferation potential with subtle differences in activity suggesting that these recombinant proteins can be used for evaluating the potency of ligands/inhibitors for targeting CXCR2-CXCL3 axis to regulate neutrophil migration.