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

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.

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

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

Amer Ashrafi, Michele Cascella, Min Kyu Cho, Farah El Turk, Bruno Claude Daniel Fauvet, Hilal Lashuel, Marilisa Neri, Hajer Ouertatani-Sakouhi, Florence Pojer, Ursula Röthlisberger, Magdalena Zweckstetter

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.

Public Library of Science2012

Elucidating the role of post-translational modifications of alpha-synuclein using semisynthesis

Mirva Hejjaoui

Alpha-synuclein (α-syn) is a natively unfolded protein that is closely linked to Parkinson’s disease (PD) by genetic, neuropathologic and biochemical evidence. Aggregated and fibrillar forms of α-syn are the main components of intracellular protein inclusions found in PD patients’ brains, termed Lewy Bodies (LB). Both in animal models and in vitro, α-syn forms fibrillar aggregates that resemble those observed in PD brain tissues. Although disease-associated mutations have been shown to promote the fibrillization of α-syn, the exact mechanisms responsible for triggering α-syn aggregation and toxicity in sporadic PD remain unknown. Addressing this gap of knowledge is crucial for understanding the molecular basis of the disease and developing effective therapies for the treatment of PD and other synucleinopathies. This project was initiated on the basis of the working hypothesis that post-translational modifications (PTM) may play important roles in modulating α-syn function and/or regulating its aggregation and toxicity. More specifically, α-syn is ubiquitously N-terminally acetylated, and phosphorylated (serines 87 and S129), ubiquitinated (lysines 12, 21 and 23) and truncated forms of α-syn have been observed in association with wild-type α-syn in LB and in brain tissues from PD patients and transgenic animals. Other modifications, such as phosphorylation at tyrosine 125 (Y125), were significantly reduced in diseased brains. Despite the discovery of candidate enzymes that mediate α-syn phosphorylation, ubiquitination and truncation, little is known about how each of these modifications alters α-syn structure, function, aggregation and toxicity in vivo. This is primarily due to the lack of tools that allow site-specific introduction of these modifications and the lack of natural mutations that can mimic the effect of these modifications, including phosphorylation. The primary focus of this thesis was to develop strategies to overcome these limitations so as to elucidate the effect of phosphorylation at Y125 and monoubiquitination at lysine 6 (K6) on α-syn structure, fibril formation, membrane binding and subcellular localization. Towards this goal, we developed two semisynthetic strategies that allow site-specific introduction of PTM in α-syn based on the ligation of synthetic peptides containing the desired modified amino acid with recombinantly expressed proteins using Expressed Protein Ligation. This approach enables the introduction of single or multiple PTM in the N- or C-terminal regions of α-syn, and the preparation of modified α-syn in milligram quantities. Using these approaches, we were able to show for the first time that ubiquitination stabilizes the monomeric form of the protein and inhibits, rather than promotes, α-syn aggregation, while phosphorylation at Y125 does not significantly change the structure and aggregation propensity of α-syn. With the semisynthetic pY125 α-syn in our hands, we were also able to investigate for the first time the sub-cellular localization of pY125 α-syn through its microinjection into primary neurons. This was not previously possible due to technical limitations related to the absence of appropriate antibodies against pY125 α-syn for immunocytochemical studies and difficulties in generating sitespecifically modified α-syn. Furthermore, we were able to investigate the effect of α-syn phosphorylation on its interactions with other proteins, by probing the effect of pS129 and pY125 on the binding to a nanobody that was specifically selected by phage-display to tightly bind to the C-terminal domain of α-syn. Accordingly, we demonstrated that phosphorylation at a single residue is capable of disrupting the binding of full-length α-syn to another protein. These results have wide-ranging implications for the potential role of phosphorylation and other PTM in regulating α-syn’s function(s). We also exploited our ability to combine semisynthetic and enzymatic approaches to investigate potential cross-talk between different PTM, namely phosphorylation at Y125 and S129 and monoubiquitination at K6. These advances would eventually allow investigating the effect of cross-talk between other N and C-terminal modifications on α-syn’s properties and to investigate PTM-dependent protein-protein and protein-ligand interactions in vitro and in cellular models of synucleinopathies. Together, our semisynthetic approaches provide novel means for the introduction of site-specific modifications in the N- and C-termini of α-syn. Current efforts in our laboratory are focused on extending these approaches to investigate the dynamics of PTM through the use of photocaged modified amino acids and to prepare novel fluorescently labeled α-syn variants to investigate its folding at the single-molecule level in living cells. The results presented in this thesis and preliminary studies from our group demonstrate that semisynthetic α-syn can provide unique opportunities to investigate structure-function of α-syn and the role of PTM in the biology of α-syn in health and disease.

EPFL2012