Characterization of Molecular Determinants of the Conformational Stability of Macrophage Migration Inhibitory Factor: Leucine 46 Hydrophobic Pocket

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 beta-strand beta 3 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.

The conformational flexibility of the carboxy terminal residues 105-114 is a key modulator of the catalytic activity and stability of macrophage migration inhibitory factor

Michele Cascella, Farah El Turk, Hilal Lashuel, Hajer Ouertatani-Sakouhi, Ursula Röthlisberger, Magdalena Zweckstetter

Macrophage migration inhibitory factor (MIF) is a multifunctional protein and a major mediator of innate immunity. Although X-ray crystallography revealed that MIF exists as a homotrimer, its oligomerization state in vivo and the factors governing its oligomerization and stability remain poorly understood. The C-terminal region of MIF is highly conserved and participates in several intramolecular interactions that suggest a role in modulating the stability and biochemical activity of MIF. To determine the importance of these interactions, point mutations (A48P, L46A), insertions (P107) at the monomer-monomer interfaces, and C-terminal deletion (Delta 110-114NSTFA and Delta 105-114NVGWNNSTFA) variants were designed and their structural properties, thermodynamic stability, oligomerization state, catalytic activity and receptor binding were characterized using a battery of biophysical methods. The C-terminal deletion mutants DeltaC5 huMIF 1-109 and DeltaC10 huMIF 1-104 were enzymatically inactive and thermodynamically less stable than wild type MIF. Analytical ultracentrifugation studies demonstrate that both C-terminal mutants sediment as trimers and exhibit similar binding to CD74 as the wild type protein. Disrupting the conformation of the C-terminal region 105-114 and increasing its conformational flexibility through the insertion of a proline residue at position 107 was sufficient to reproduce the structural, biochemical and thermodynamic properties of the deletion mutants. P107 MIF forms an enzymatically inactive trimer and exhibits reduced thermodynamic stability relative to the wild type protein. To provide a rationale for the changes induced by these mutations at the molecular level, we also performed molecular dynamics simulations on these mutants in comparison to the wild type MIF. Together, our studies demonstrate that intersubunit interactions involving the C-terminal region 105-114, including a salt-bridge interaction between Arg73 of one monomer and the carboxy terminus of a neighboring monomer, play critical roles in modulating tertiary structure stabilization, enzymatic activity, and thermodynamic stability of MIF, but not its oligomerization state and receptor binding properties. Our results suggest that targeting the C-terminal region could provide new strategies for allosteric modulation of MIF enzymatic activity and the development of novel inhibitors of MIF tautomerase activity.

American Chemical Society2008

Elucidating the Role of Quaternary Structure in Modulating Macrophage Migration Inhibitory Factor (MIF) Functions

Farah El Turk

Macrophage Migration Inhibitory Factor (MIF) is a multifunctional protein and a major mediator of innate immunity. The paramount importance of MIF in the pathogenesis of many inflammatory, infectious, auto-immune and oncogenic diseases led to its emergence as a viable therapeutic target. Understanding MIF' biological and structural properties is of fundamental importance to provide insights into the molecular bases underlying its mode of action, and to develop effective strategies to diagnose, prevent or treat MIF associated diseases. Although X-ray crystallography revealed that MIF exists as a homotrimer, its oligomerization state in vivo as well as the factors governing its oligomerization and stability remain poorly understood. In order to assess the structural components crucial for MIF's quaternary structure, conformational stability and function, we sought to target key interactions within MIF trimer via genetic and biochemical tools. To achieve this goal, we employed a battery of computational, biochemical, biophysical and structural biology approaches. Point mutations, insertions, and deletions MIF variants, designed in silico at the monomer-monomer interfaces of MIF's X-ray structure, were generated, expressed and characterized using a battery of biophysical methods, in vitro. In silico and molecular dynamic studies performed on MIF's X-ray structure, allowed identifying several regions crucial for MIF's structure. First, our biophysical studies demonstrate that inter-subunit interactions involving the C-terminal region 105-114 play critical roles in modulating tertiary structure stabilization, enzymatic activity, and thermodynamic stability of MIF, but not its oligomerization state and receptor binding properties (Chapter 1). Carboxy-terminus truncated mutants (ΔC5 huMIF1-109 and ΔC10 huMIF1-104), as well as the insertion-mutant P107 MIF (designed to increase the flexibility of the C-terminal region) form enzymatically inactive trimers and exhibit reduced thermodynamic stability relative to the wild Type protein. On the other hand, our results suggest that targeting the C-terminal region could provide new strategies for allosteric modulation of MIF enzymatic activity and the development of novel inhibitors of MIF tautomerase activity. We also demonstrated that a stable MIF monomeric state does not populate in vitro as the monomer undergoes rapid aggregation upon dissociation from the trimer. Furthermore, we described and characterized novel intersubunit interactions, involving the packing of a Leu46 side chain from one monomer into a hydrophobic pocket from the adjacent monomer (Chapter 2). Our findings provide new insight into the role of this pocket in modulating conformational states of MIF in solution. Also, disrupting this hydrophobic interaction through point mutations (L46G, L46A and L46F), is sufficient to decrease MIF stability to modulate its catalytic activity through tuning of the enzyme's efficiency and affinity towards its substrate. In the final chapter of this theses (Chapter 3), we describe our efforts to generate an in silico protocol for virtual high throughput screening of large libraries of small molecules and the discovery of novel inhibitors of MIF targeting its tautomerase activity. Our protocol takes into account the molecular dynamic parameters of MIF and aims at combined 1Docking standalone (Surflex scoring function), 2Docking (FlexX scoring function) coupled to Molecular Dynamics Simulations (MDS), and a 3combination of Docking score from FlexX coupled to MDS and Pharmacophore Filtering. in vitro validation of our virtual protocol led to the identification of three news inhibitors of MIF, with IC50 lower than 5 µM. Together, our studies provide i) new insights into structural properties of MIF and factors governing its oligomerization; ii) novel molecular tools (MIF mutants and small molecules) that can be used to dissect the structure-function relationship of MIF in health and disease; and iii) novel technical tool to identify small molecules inhibitors of MIF. Together, these results should contribute to the development of structure-based therapeutic strategies targeting specific molecular pathway(s), or structural aspect(s) relevant for its functions and role in health and diseases.

EPFL2010

A new class of isothiocyanate-based irreversible inhibitors of macrophage migration inhibitory factor

Farah El Turk, Bruno Claude Daniel Fauvet, Hilal Lashuel, Hajer Ouertatani-Sakouhi, Magdalena Zweckstetter

Macrophage migration inhibitory factor (MIF) is a homotrimeric multifunctional proinflammatory cytokine that has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. Current therapeutic strategies for targeting MIF focus on developing inhibitors of its tautomerase activity or modulating its biological activities using anti-MIF neutralizing antibodies. Herein we report a new class of isothiocyanate (ITC)-based irreversible inhibitors of MIF. Modification by benzyl isothiocyanate (BITC) and related analogues occurred at the N-terminal catalytic proline residue without any effect on the oligomerization state of MIF. Different alkyl and arylalkyl ITCs modified MIF with nearly the same efficiency as BITC. To elucidate the mechanism of action, we performed detailed biochemical, biophysical, and structural studies to determine the effect of BITC and its analogues on the conformational state, quaternary structure, catalytic activity, receptor binding, and biological activity of MIF. Light scattering, analytical ultracentrifugation, and NMR studies on unmodified and ITC-modified MIF demonstrated that modification of Pro1 alters the tertiary, but not the secondary or quaternary, structure of the trimer without affecting its thermodynamic stability. BITC induced drastic effects on the tertiary structure of MIF, in particular residues that cluster around Pro1 and constitute the tautomerase active site. These changes in tertiary structure and the loss of catalytic activity translated into a reduction in MIF receptor binding activity, MIF-mediated glucocorticoid overriding, and MIF-induced Akt phosphorylation. Together, these findings highlight the role of tertiary structure in modulating the biochemical and biological activities of MIF and present new opportunities for modulating MIF biological activities in vivo.

American Chemical Society2009