Granulocyte macrophage colony stimulating factor (GMCSF) is a cytokine that is involved in myelopoiesis as well as immunomodulation. GMCSF has been shown to worsen conditions such as rheumatoid arthritis (RA) and multiple sclerosis (MS) while ameliorating diseases such as Type I Diabetes. GMCSF has also been used clinically to combat neutropenia based on its immunostimulatory activity. Pathophysiological properties of GMCSF have been highlighted by the incidence of the global pandemic, COVID-19 (caused by SARS-CoV-2), where elevated levels of GMCSF were identified in COVID-19 patients in cases related to development of systemic hyperinflammation, pneumonia and acute respiratory distress syndrome (ARDS). A key feature of signalling molecules like GMCSF is the selective and nuanced way in which responses can be tuned to external stimuli. The mechanism of how these signals are modulated are unknown and of particular interest. GMCSF and heparin have been shown to interact under low pH conditions. This binding interaction is important in two ways. Firstly, this pH sensitive interaction can serve as a basis to understand how cytokines can selectively interact with binding partners under variable physiological conditions. Secondly, in clinical settings where both anticoagulatory and immunostimulatory drugs are required, clear understanding of the binding interactions between the two drugs will help inform treatment decisions. Previously we identified heparin dependent structural changes as well as a striking dynamic change in GMCSF that was dependent on heparin chain length and pH using solution NMR studies which we believe to be dependent on a histidine triad. Here we have characterized GMCSF mutants using sited-directed mutagenesis to exchange critical histidine residues for alternate amino acid residues (including Ala, Phe, Tyr and Asp) to probe the contributions of histidine residues to the increased flexibility of GMCSF observed in acidic conditions. We find that the histidine residues are critical for stability and as little as one amino acid difference will alter flexibility and stability of the protein. In some instances, mutations of all three histidine residues can lead to partial protein unfolding. Our results highlight the critical chemical properties at the amino acid level that allow GMCSF to interact with heparin in an environmentally dependent way.
