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Abstract
Macrophage Migration Inhibitory Factor (MIF) is a key mediator of inflammatory responses and innate immunity and has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. The oligomerization of MIF, more specifically trimer formation, is essential for its keto-enol tautomerase activity and probably mediates several of its interactions and biological activities, including its binding to its receptor CD74 and activation of certain signaling pathways. Therefore, understanding the molecular factors governing the oligomerization of MIF and the role of quaternary structure in modulating its structural stability and multifunctional properties is crucial for understanding the function of MIF in health and disease. Herein, we describe highly conserved intersubunit interactions involving the hydrophobic packing of the side chain of Leu46 onto the β-strand β3 of one monomer within a hydrophobic pocket from the adjacent monomer constituted by residues Arg11, Val14, Phe18, Leu19, Val39, His40, Val41, Val42, and Pro43. To elucidate the structural significance of these intersubunit interactions and their relative contribution to MIF’s trimerization, structural stability and catalytic activity, we generated three point mutations where Leu46 was replaced by glycine (L46G), alanine (L46A) and phenylalanine (L46F), and their structural properties, stability, oligomerization state, and catalytic activity were characterized using a battery of biophysical methods and X-ray crystallography. Our findings provide new insights into the role of the Leu46 hydrophobic pocket in stabilizing the conformational state of MIF in solution. Disrupting the Leu46 hydrophobic interaction perturbs the secondary and tertiary structure of the protein but has no effect on its oligomerization state.
Highlights
Macrophage Migration Inhibitory Factor (MIF) is a ubiquitous multifunctional protein and a key player in the inflammatory response and innate immunity
During the last two decades MIF has been shown to be involved in a wide range of cellular processes, e.g. transcriptional regulation of inflammatory gene products [3], cell cycle control [4,5], modulation of cell proliferation and differentiation [6], regulating glucocorticoıd activity [7], inactivation of p53 tumor suppressor factor [8] and signal transduction, and emerged as an important player in the molecular mechanisms underlying the pathogenesis of several inflammatory autoimmune diseases including arthritis [9,10,11], multiple sclerosis [12,13], diabetes [14], sepsis [15,16,17], atherosclerosis [18] and oncogenesis [19,20,21,22,23,24,25]
Thermal unfolding monitored by far UV Circular Dichroism (CD) at 218 nm demonstrated that disruption of leucine 46 (Leu46) hydrophobic site induces a clear destabilization of MIF structure stability (Figure 2F)
Summary
Macrophage Migration Inhibitory Factor (MIF) is a ubiquitous multifunctional protein and a key player in the inflammatory response and innate immunity. During the last two decades MIF has been shown to be involved in a wide range of cellular processes, e.g. transcriptional regulation of inflammatory gene products [3], cell cycle control [4,5], modulation of cell proliferation and differentiation [6], regulating glucocorticoıd activity [7], inactivation of p53 tumor suppressor factor [8] and signal transduction, and emerged as an important player in the molecular mechanisms underlying the pathogenesis of several inflammatory autoimmune diseases including arthritis [9,10,11], multiple sclerosis [12,13], diabetes [14], sepsis [15,16,17], atherosclerosis [18] and oncogenesis [19,20,21,22,23,24,25]. The physiological relevance of these activities and their role in regulating the function of MIF in health and disease remain controversial [33,34]; the physiological substrates for both catalytic activities are yet to be discovered
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