title: Characterization of molecular determinants of the conformational stability of macrophage migration inhibitory factor: leucine 46 hydrophobic pocket.
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.
authors.affiliation: Laboratory of Molecular and Chemical Biology of Neurodegeneration, Department of Life Sciences, Swiss Federal Institute of Technology, Lausanne, Switzerland.
authors.forename: Farah
authors.initials: F
authors.lastname: El-Turk
authors.forename: Bruno
authors.initials: B
authors.lastname: Fauvet
authors.forename: Amer
authors.initials: A
authors.lastname: Ashrafi
authors.forename: Hajer
authors.initials: H
authors.lastname: Ouertatani-Sakouhi
authors.forename: Min-Kyu
authors.initials: MK
authors.lastname: Cho
authors.forename: Marilisa
authors.initials: M
authors.lastname: Neri
authors.forename: Michele
authors.initials: M
authors.lastname: Cascella
authors.forename: Ursula
authors.initials: U
authors.lastname: Rothlisberger
authors.forename: Florence
authors.initials: F
authors.lastname: Pojer
authors.forename: Markus
authors.initials: M
authors.lastname: Zweckstetter
authors.forename: Hilal
authors.initials: H
authors.lastname: Lashuel
mesh_terms: D000595:Amino Acid Sequence; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D019746:Intramolecular Oxidoreductases / Q000737:chemistry*; D007930:Leucine / Q000737:chemistry*; D008263:Macrophage Migration-Inhibitory Factors / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry; D009154:Mutation / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D055503:Protein Multimerization; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship
chemical_list: D008263:Macrophage Migration-Inhibitory Factors; D050505:Mutant Proteins; D019746:Intramolecular Oxidoreductases; C515598:MIF protein, human; D007930:Leucine