Molecular characterization of allosteric signaling in D-dopachrome tautomerase

Chen, Emily

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Year:: 2022
Contributor:: Chen, Emily (creator); Jogl, Gerwald (Reader); Fawzi, Nicolas (Reader); Monaghan, Sean (Reader); Smith, Colin (Reader); Lisi, George (Advisor); Brown University. Department of Molecular Biology, Cell Biology and Biochemistry (sponsor)
Genre:: theses
Subject:: Nuclear magnetic resonance spectroscopy; Structural biology; Allostery; Macrophage migration inhibitory factor
Extent:: , None p.

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Abstract:: Macrophage migration inhibitory factor (MIF) and its homolog MIF-2 are cytokine-like proteins with critical roles in immunomodulation that are upregulated in various inflammatory diseases. These proteins are broadly and constitutively expressed and bind CD74 to induce the expression of pro-inflammatory cytokines, initiate ERK1/2 signaling, and to recruit leukocytes. Commercial inhibitors of MIF target an enzymatic site, which has been demonstrated to alter CD74-mediated activity when modified by mutation and inhibitor binding. However, their therapeutic efficacy is hindered by a lack of molecular-level characterization of the effects of inhibitor binding. A dynamic relay connecting the MIF catalytic site to an allosteric site at Tyr99 was previously reported. Its connectivity to the N- and C-terminus was determined by molecular simulations and confirmed by experimentation where mutation at this site altered enzymatic activity and CD74 activation. The structural conservation and functional overlap of MIF and MIF-2 prompted the question of whether this dynamic regulatory network is conserved in MIF-2. Nuclear magnetic resonance (NMR) spectroscopy, tautomerase assays, and circular dichroism was used in combination with X-ray crystallography, in vivo mouse models, and molecular simulations from collaborators to present the structural, dynamic, and functional consequences of mutating allosteric residues. Chapter Two details the N-terminal portion from Pro1 to Phe100, the allosteric site in MIF-2. Chapter Three investigates the roles of Ser62 and Phe100 in the network. Chapter Four defines the C-terminal segment continuing from Phe100 to proposed CD74-activating residues based on homology to MIF. In each chapter, mutations of residues in the allosteric network caused a range of structural and dynamic perturbations such that flexibility correlated negatively with enzymatic activity. Further, mutations at the N-terminus result in structural and dynamic perturbations at the C-terminus, and vice versa, demonstrating allosteric reciprocity. This thesis establishes that dynamic communication is preserved in MIF-2 despite differences in primary sequence, defining a complete network from the N-terminal enzymatic site to C-terminal CD74-activating residues that can be methodically targeted for functional control of MIF family-induced inflammation.
Notes:: Thesis (Ph. D.)--Brown University, 2022

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Chen, Emily, "Molecular characterization of allosteric signaling in D-dopachrome tautomerase" (2022). Molecular Biology, Cell Biology, and Biochemistry Theses and Dissertations. Brown Digital Repository. Brown University Library. https://repository.library.brown.edu/studio/item/bdr:vz3cka2e/

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Molecular Biology, Cell Biology, and Biochemistry Theses and Dissertations

Theses and Dissertations for the Molecular Biology, Cell Biology, and Biochemistry department.
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