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A Cysteine Variant at an Allosteric Site Alters MIF Dynamics and Biological Function in Homo- and Heterotrimeric

Erin Skeens1, Georgios Pantouris2,3, Dilip Shah4

  • 1Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI, United States.

Frontiers in Molecular Biosciences
|March 7, 2022
PubMed

Insights

Investigating Macrophage Migration Inhibitory Factor (MIF) allosteric site mutations, this study characterizes the Tyr99Cys (Y99C) variant. The research provides insights into MIF

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • Macrophage Migration Inhibitory Factor (MIF) is a conserved inflammatory protein with diverse functions.
  • Human MIF functions as a homotrimer, possesses enzymatic activity, and interacts with receptors like CD74.
  • A solvent channel and an allosteric gating residue regulate MIF's enzymatic activity and receptor binding.

Purpose of the Study:

  • To investigate the functional consequences of mutating the allosteric residue Tyr99 to cysteine (Y99C) in human MIF.
  • To characterize the structural, dynamic, and functional properties of the Y99C MIF variant.
  • To explore the allosteric regulation of MIF by creating and analyzing heterotrimeric variants.

Main Methods:

  • Site-directed mutagenesis to create the Y99C MIF variant.
  • X-ray crystallography to determine the protein's structure.
  • Nuclear Magnetic Resonance (NMR) spectroscopy to analyze protein dynamics.
  • Biochemical assays to assess enzymatic activity and CD74 binding.

Main Results:

  • The Y99C mutation was introduced, and the crystallographic structure of the variant was determined.
  • NMR dynamics revealed changes associated with the allosteric site modification.
  • Characterization of CD74 function and enzymatic activity of the Y99C variant was performed.
  • Strategies for generating and purifying heterotrimeric MIF variants were developed.

Conclusions:

  • The Y99C mutation impacts MIF's structural dynamics and allosteric regulation.
  • Understanding the Y99C variant provides insights into MIF's catalytic mechanism and CD74 interactions.
  • This research facilitates further studies on MIF allosteric modulation and therapeutic targeting.
Keywords:
allosteric sitebiological activitiesbiophysicsmacrophage migration inhibitory factormutagenesis

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