Role of the Cysteine 81 Residue of Macrophage Migration Inhibitory

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Role of cysteine 81 residue of Macrophage Migration Inhibitory Factor as a molecular redox switch Alexander Schinagl, Randolf J Kerschbaumer, Nicolas Sabarth, Patrice Douillard, Peter Scholz, Dirk Voelkel, Julia Hollerweger, Peter Goettig, Hans Brandstetter, Friedrich Scheiflinger, and Michael Thiele Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b01156 • Publication Date (Web): 07 Feb 2018 Downloaded from http://pubs.acs.org on February 11, 2018

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Biochemistry

Role of cysteine 81 residue of Macrophage Migration Inhibitory Factor as a molecular redox switch Alexander Schinagl†, Randolf J. Kerschbaumer†, Nicolas Sabarth†, Patrice Douillard†, Peter Scholz†, Dirk Voelkel†, Julia C. Hollerweger‡, Peter Goettig‡, Hans Brandstetter‡, Friedrich Scheiflinger†*, Michael Thiele†* † Baxalta Innovations GmbH, Uferstrasse 15, 2304 Orth a. d. Donau, Austria ‡ Division of Structural Biology and Bioinformatics, University of Salzburg, Billrothstr. 11, 5020 Salzburg, Austria *Corresponding authors Keywords: MIF, cysteine 81, redox switch, anti-oxMIF antibodies

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ABSTRACT Macrophage migration inhibitory factor (MIF) is a proinflammatory and tumor promoting cytokine that occurs in two redox-dependent immunologically distinct conformational isoforms. The disease-related structural isoform of MIF (oxMIF) can be specifically and predominantly detected in the circulation of patients with inflammatory diseases and in tumor tissue, whereas the ubiquitously expressed isoform of MIF (redMIF) is abundantly expressed in healthy as well as in diseased subjects. In this article we report that cysteine 81 within MIF serves as a “switch cysteine” for the conversion of redMIF to oxMIF. Modulating cysteine 81 by thiol-reactive agents leads to significant structural rearrangements of the protein, resulting in a reduced β-sheet and increased random coil content, but maintaining the trimeric quaternary structure. This conformational change in the MIF molecule enables binding of oxMIF-specific antibodies BaxB01 and BaxM159, which showed beneficial activity in animal models of inflammation and cancer. Crystal structure analysis of the MIF-derived EPCALCS peptide, bound in its oxMIF-like conformation by the Fab fragment of BaxB01, revealed that this peptide adapts a curved conformation, making the central thiol protein oxidoreductase motif competent to undergo disulfide shuffling. We conclude that redMIF might reflect a latent zymogenic form of MIF and formation of oxMIF leads to a physiologically relevant, i.e. enzymatically active state.


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INTRODUCTION Macrophage migration inhibitory factor (MIF) was one of the first cytokine-like molecules to be investigated and was discovered more than 50 years ago. Unlike other pro-inflammatory cytokines, MIF is expressed broadly among cell types in cytoplasmic pools1 and is secreted through a non-canonical pathway2 after cellular exposure to various stimulants such as TLR antigens, mitogens, and pro-inflammatory cytokines3. MIF is constantly present in human plasma at levels of up to 20 ng/ml4, 5 and furthermore stored intracellularly in many tissues. MIF exerts its biological activity via receptor and non-receptor interactions and was reported to stimulate cell proliferation and differentiation by ERK1/2 activation, contribute to cell survival by activation of the PI3K/Akt pathway and inhibition of tumor suppressor p53, and to induce cell migration and expression of other inflammatory mediators6-8. Moreover, MIF acts as a potent angiogenic factor in autoimmune diseases and cancer, by inducing angiogenic mediators like vascular endothelial growth factor (VEGF) and IL-89, 10. MIF has two enzymatic activities: a dopachrome tautomerase activity11,

12

located at the N-

terminus and a thiol- protein oxidoreductase activity (TPOR) activity conferred by a CXXC motive in the center of the molecule13. Despite substantial evidence on the importance of the tautomerase activity of MIF in vitro, its physiological role has been discussed controversially, as no relevant physiological substrate has yet been identified11,

12, 14-16

. Similarly, MIF exhibits

catalytic TPOR activity in vitro, acting primarily as a disulfide reductase, but since today no physiological substrate has been identified13. In addition to the CXXC cysteine residues at positions 57 (C57) and 60 (C60), MIF has a third cysteine at position 81 (C81). The catalytic TPOR activity is fully dependent on the presence of C60, whereas the C57 mutant shows partial


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activity. Studies with a MIF(C81S) mutant suggest that this residue is not involved in the oxidoreductase activity of MIF13, 17. We recently reported that MIF occurs in two immunologically distinct redox-dependent isoforms, termed oxidized MIF (oxMIF) and reduced MIF (redMIF)18. RedMIF was found to be the abundantly expressed isoform of MIF that is present in healthy and diseased subjects. In contrast, oxMIF represents the disease-related isoform which was detected predominantly in cancerous tissue and in the circulation and on the surface of immune cells of patients with inflammatory diseases18, 19. We furthermore described human monoclonal antibodies BaxB01 and BaxM159 which were shown to be highly specific for oxMIF, but did not recognize redMIF. These oxMIF specific antibodies neutralize MIF functional activity in cell-based assays and were shown to be protective in animal models of inflammation and cancer18, 20, 21. Antibodies that were not able to discriminate between MIF and oxMIF did not show protective effects in vivo

20

. Conversion of

redMIF, the ubiquitous form of MIF, to oxMIF, the disease related isoform of MIF, occurs during disease progression and can be mimicked in vitro by oxidation of E.coli derived recombinant human MIF with either L-cystine or oxidized glutathione (GSSG). However the molecular basis for the structural change of redMIF to oxMIF was unknown so far. Here we report that C81 of MIF serves as a ”key switch” amino acid which acts as a donor of reducing equivalent and is essential in the conversion of MIF into its disease related isoform, oxMIF.


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Biochemistry

MATERIALS AND METHODS Reagents Recombinant MIF, BaxB01, BaxG03, BaxM159 and an isotype-matched human control antibody were produced as described20. OxMIF (surrogate oxMIF) was prepared as follows: Cys-MIF: 40 µM MIF in PBS was incubated with 3-fold excess (volume) of a saturated L-Cystine solution for 2-4 h at 37°C; DTNB-MIF: 100µM MIF in 100 mM sodium phosphate buffer with 1 mM EDTA at pH 8.0 (reaction buffer) was incubated with a 3-fold molar excess of DTNB (in reaction buffer) for 16h at 2-8°C; EMCA and MMTS MIF: 100 µM MIF in PBS with 5 mM EDTA was incubated with a 3-fold molar excess of thiol-reactive agent (DMSO Stock) for 16h at 2-8°C. All samples were desalted two times against PBS by 7K MWCO Zeba Spin Desalting Columns. Protein expression and purification Polyclonal rabbit anti-MIF antibodies were generated by immunization with recombinant MIF and purified from serum over protein A columns (GE Healthcare) and immobilized recombinant MIF. Recombinant wild-type (wt) (UniProt P14174), and cysteine to serine mutant fusion constructs of human MIF (huMIF) with streptavidin binding peptide (SBP) tag and (GGGGS)2linker and cysteine 81 mutants were cloned and expressed in E.coli Shuffle T7 Express (New England Biolabs) by standard methods. Bacterial pellets were harvested by centrifugation and soluble protein was extracted using BugBuster® Protein Extraction Master Mix (Novagen), including cOmplete™ Mini EDTA-free Protease Inhibitor Cocktail (Roche) according to manufacturer instructions. Cell debris was removed by centrifugation and filtration. The cleared soluble extract was mixed with equal volumes of 200 mM sodium phosphate buffer, pH 7.2 with 500 mM NaCl and was purified by affinity chromatography via streptavidin columns (Thermo Scientific). Bound proteins were eluted with 0.5 mM biotin in PBS. The supernatant of human Seite 5 von 31 ACS Paragon Plus Environment

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MIF and mutants without the SBP fusion protein was directly applied to an anion exchange chromatography column (HiTrap 26/16 DEAE FF, GE Healthcare). The flow through, containing MIF, was re-buffered in 20 mM Bis/Tris pH 6.3 and further purified by a cation exchange chromatography (HiTrap SP FF, GE Healthcare). Highly pure human MIF was eluted by a salt gradient at about 50 mM NaCl in 20 mM Bis/Tris buffer, pH 6.3. Finally, the purified recombinant proteins were polished and re-buffered in PBS by size exclusion chromatography (Hi Load Superdex 75 26/60 pg, GE Healthcare). Epitope mapping Linear peptides and Chemically Linked Peptides on Scaffolds (CLIPS Pepscan B.V., NL) which structurally fixes peptides into defined three-dimensional structures like single, double or triple looped structures as well as sheet- and helix-like folds and combinations thereof were used in an ELISA format. The following peptide constructs were prepared; (i) Linear peptides with 15 amino acids and an overlap of 14 amino acids that cover the full MIF sequence. Peptides containing cysteines have also been made with a serine replacement. (ii) 20mer peptides with an overlap of 19 amino acids connected by a P2 CLIPS™, thus promoting helical conformation in the peptides, (iii) 20mer peptides with an overlap of 19 amino acids, where residues 8 and 11 are replaced by artificial cysteines, and were connected by a P2 CLIPS™, thus promoting β-sheet conformation in the peptides and (iv) 33mer peptides with discontinuous epitopes using a matrix design, with artificial cysteines introduced at positions 1, 17, 33, which were connected by a T3CLIPS™. The ranges 2 – 16 and 18 – 32 contain all 15mer peptides with an overlap of 12 amino acids, derived from the MIF sequence. The binding of antibody to each of the synthesized peptides was tested in ELISA. The immobilized peptides were incubated with oxMIF specific


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Biochemistry

antibodies (BaxB01 or BaxM159) and were detected with an anti-human IgG antibody peroxidase conjugate. Semi quantitative oxMIF ELISA OxMIF specific antibodies (BaxB01, BaxM159 and matched human isotype IgG1) were immobilized into 96-well micro plates (NUNC Maxisorp) overnight at 4°C. The plates were rinsed with TBST and blocked with 2% FG/TBST or 2% BSA/TBST (dilution buffer) for at least 1 h at room temperature. After rinsing the plates with TBST, the antibodies were incubated with recombinant human MIF/oxMIF surrogates/MIF mutants diluted in dilution buffer ± 1 mM DTT for 1-2 h at room temperature. Captured oxMIF (surrogate oxMIF) was detected with polyclonal rabbit anti-MIF antibodies and a goat anti-rabbit-IgG peroxidase conjugate. After a final rinse with TBST the plates were developed with TMB. The reaction was stopped with 3 M sulfuric acid and the plates were read in a 96-well plate reader at 450 nm. ELISA data are representative of at least three independent experiments. Mass spectrometry For mass determination in the linear mode, 1-2 µg of each sample were desalted on C4 ZipTip columns (Millipore) (equilibrated and washed with 0.1% trifluor acetic acid, eluted with 2 µl 80% acetonitrile/0.1% trifluor acetic acid). Consequently, 0.5 µl of the eluate was mixed with 0.5 µl 10 mg/ml sinapinic acid (Fluka) in 30% acetonitrile/0.1% trifluor acetic acid as sample matrix. The measurement was done using a 4800 MALDI TOF/TOF Analyzer (AB Sciex) in linear mode with a mass range of m/z 5000-30000. The raw data were imported in mMass v5.5 and the peaks were annotated according to their apparent mass.


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For peptide mass fingerprinting analysis the samples were diluted with 2 M urea/0.1 M ammonium bicarbonate, pH 8 without reduction or alkylation and digested with 0.4 µg of trypsin (Serva) at 37°C overnight. The samples were desalted using ZipTip C18 columns (Millipore) (see above). Subsequently, the eluates were mixed with α-cyano-4-hydroxycinnamic acid (Bruker) as sample matrix. The measurement was done with a 4800 MALDI TOF/TOF Analyzer (AB Sciex) in the reflector mode with a mass range of m/z 700-7000. The raw data were imported in mMass V5.5 and the peaks were annotated according to their apparent mass and compared to reference digests of the proteins with trypsin calculated in the program. Near and Far-UV Circular Dichroism (CD) Samples were diluted in their corresponding formulation buffer (either PBS or PBS containing 1 mM DTT) to a final concentration of 1 mg/ml and measured in a 10 mm or 1 mm quartz cuvette for Near- and Far-UV CD analysis respectively using a Jasco J-815 spectropolarimeter. Near-UV CD spectra (320 – 250 nm) were recorded at a scan speed of 100 nm/min, a band width of 1 nm, a response time of 2 sec, a data pitch of 0.1 nm and eight accumulations. Far-UV CD spectra (260– 190 nm) were recorded at a scan speed of 100 nm/min, a band width of 1 nm, a response time of 2 sec, a data pitch of 0.1 nm and 8 accumulations. For secondary structure estimation, the spectra were analyzed by using a CDPro and the CONTIN algorithm. The estimations were carried out via two different reference databases, namely SDP48 and SMP56. The reference database leading to the best fit with the lowest relative mean standard deviation was chosen for comparison of all samples. SV-AUC For SV-AUC samples were analyzed in a ProteomeLab XL-A analytical ultracentrifuge (Beckman-Coulter) at 20 °C, using a 4-hole An-60 Ti analytical rotor and 12-mm epon-charcoal Seite 8 von 31 ACS Paragon Plus Environment

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Biochemistry

double-sector centerpieces with sapphire windows. Rotor speed optimization for each sample was performed at 40,000 rpm and 50,000 rpm, respectively. The evolution of sedimentation profiles at 40 and 50 krpm, respectively, was recorded by using absorbance detection optics at a wavelength of 280 nm. Data analysis for samples was performed with Sedfit (Version 15.01b). SV-AUC raw data was pre-processed (meniscus position fixed, bottom position fit, and time-invariant and radially invariant noise correction) and analyzed by using the continuous c(s) distribution model with Tikhonov maximum entropy regularization (confidence level: 0.95) for the assessment of sedimentation coefficient c(s) and molecular weight distributions c(M), respectively. All sedimentation coefficients are reported as corrected values for water at 20 °C. Protein crystallography Crystals of BaxB01-Fab grew in in 100 mM Tris/HCl, pH 6.4, 25% (w/v) PEG 3350, while complex formation of BaxB01-Fab with the MIF derived peptide EPCALCS was achieved by incubation with a 1.5-fold molar excess of the peptide EPCALCS or the seleno-cysteine containing EPCALSecS (99 mM stock solution in DMSO) for 2 hours at 4 °C. Afterwards crystallization of the complex took place in 100 mM Tris/HCl pH 8.0, 100 mM MgAc2 and 12% PEG-MME 5500. Large crystals of both types were obtained by mixing of protein solution with crystallization buffer, equilibrated against reservoir solution at 20 °C. Data collection of crystals frozen in liquid nitrogen (100 K) was conducted at the ID30A-3 beamline (ESRF, Grenoble). Indexing and integration of acquired datasets was performed with iMOSFLM32, subsequent scaling and merging with SCALA33, both part of the program suite CCP434. A Fab fragment from PDB entry 4Q9Q without aptamer was used for phasing in a molecular replacement approach with the PHASER software, yielding a solution for BaxB01-Fab with two molecules in the asymmetric unit (asu) in space group P41212 at 2.6 Å resolution35. For the BaxB01-FabSeite 9 von 31 ACS Paragon Plus Environment

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EPCALCS complex, the previously determined structure of Baxb01-Fab was utilized for molecular replacement, resulting in a solution with 24 molecules in the asu in space group C2 at 4.0 Å resolution. In order to circumvent model bias, SFCHECK36 was utilized to calculate omit maps. Anomalous diffraction of the Baxb01-Fab-EPCALSecS complex at the selenium peak wavelength allowed locating the position of the seleno-cysteine sidechains. Models were built in COOT37 by iterative cycles of structure refinement by REFMAC538 and PHENIX39. Structural figures were prepared with PyMol (PyMOL Molecular Graphics System, Version 1.7rc1, Schrödinger, LLC, 2014). The crystallographic R values used in table 3 are defined as R=∑hkl∑j|Ihkl,j−⟨Ihkl⟩|∑hkl∑jIhkl,j, where ⟨Ihkl⟩ is the average of symmetry-related observations of a unique reflection. The Rp.i.m. takes the improved precision of the average intensities

resulting

from

the

multiplicity

of

the

measurements

into

account:

Rp.i.m.=∑hkl1n−1√∑nj=1|Ihkl,j−⟨Ihkl⟩|∑hkl∑jIhkl,j.

RESULTS Antibodies specific for oxMIF recognize linear epitopes that are buried according to the crystal structure of redMIF. Elucidation of the crystal structure of MIF revealed that it forms a barrel shaped homotrimer22-24. Each monomer of MIF contains two antiparallel α-helices and six β-strands (Figure 1A). Four of the six β-strands form a β-sheet, which forms a solvent-accessible central channel in the MIF trimer. The remaining two β-strands have been shown to interact with the β sheets of adjacent MIF subunits in a trimer. However, MIF that was used for the reported crystallography studies was derived from E.coli without an oxidative step and therefore corresponds to redMIF. The oxMIF specific antibodies BaxB01 and BaxM159 target regions of the MIF molecule which are Seite 10 von 31 ACS Paragon Plus Environment

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Biochemistry

located in the central core of the MIF trimer which might not be directly accessible. We confirmed the binding region and identified the precise epitope by using immobilized linear 15mer peptides with an overlap of 14 amino acids that cover the full MIF sequence which were probed with BaxB01 and BaxM159 (Figure 1B). We identified the shortened linear epitopes P56CALC60 and D93RVYINYY100 for BaxB01 and BaxM159 respectively. By replacing each amino acid by serine in the 15mer peptides containing the full linear epitopes of these antibodies, we discovered the essential amino acids in the short linear epitopes; P57, C57 and C60 for BaxB01 and D93, Y96, Y99, Y100 for BaxM159 (Figure 1C). In addition, we aimed to elucidate potential non-linear epitopes using the CLIPS Epitope Mapping technology. However, neither single, double or triple looped structures nor sheet- and helix-like folds nor discontinuous structure linked peptide epitopes could be identified for both antibodies (data not shown). The refined epitopes of BaxB01 (aa 56-60) and BaxM159 (aa 93-100) are illustrated in the ribbon structure of the MIF monomer (Figure 1A) and in a surface rendering of the MIF trimer (Figure 1D-F). From this illustration it is evident that only a few amino acid residues from the peptides spanning the antibody epitopes are facing towards the surface of the MIF trimer. Thus, we postulated that a major structural rearrangement in the redMIF molecule is required to enable binding of the anti-oxMIF antibodies. Previous experiments have demonstrated that the structural rearrangement can be induced in vitro by mild oxidation18. Hence, we sought to investigate the contribution of the three cysteines to the conversion of redMIF to oxMIF.


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Figure 1 Epitope mapping, ribbon structure and surface rendering of MIF with epitopes of anti-oxMIF antibodies BaxB01 and BaxM159. (A) Ribbon structure of MIF monomer (PDB, 3DJH chain A from) depicting the refined linear epitopes of BaxB01 (aa 56-60, red) and BaxM159 (aa 93-100, blue). (B) Epitope mapping using linear 15mer peptides with an overlap of 14 amino acids that cover the full MIF sequence which were probed with either BaxB01 (red), or BaxM159 (blue). (C) 15mer peptides with subsequent exchange of amino acids by serine to identify the essential epitopes of BaxB01 (red, left panel) and BaxM159 (blue, right panel). (D-F) Surface rendering (using PyMol, PDB 3DJH) of the 3-D crystal structure of MIF (D, top view; E, bottom view and F, side view) with the epitopes of BaxB01 (red) and BaxM159 (blue) and A, B and C chain of MIF shaded in light grey, grey and dark grey respectively.

Mild oxidation enables binding of oxMIF specific antibodies by chemical modification of C81. To investigate which modification of MIF leads to the conversion to oxMIF we used prooxidative reagents that selectively target free reactive cysteines. The sulfhydryl reactive compounds EMCA, DTNB and MMTS which introduce side groups with different charge, polarity and size, as well as L-cystine were selected to generate oxMIF-derivatives. EMCA, which forms covalent thioether bonds with free thiols, did not induce a structural change of MIF Seite 12 von 31 ACS Paragon Plus Environment

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Biochemistry

sufficient to allow strong binding of anti-oxMIF mAbs BaxB01 and BaxM159 (Figure 2A). In contrast, L-cystine, MMTS and DTNB, which form covalent mixed disulfides with free thiols, induced a structural rearrangement in the MIF molecule leading to a significant binding to BaxB01 and BaxM159 (Figure 2A). Among the reagents used, DTNB introduced the largest side group (aromatic benzoic ring structure), as well as a highly negatively charged carboxyl group and a nitric oxide group and DNTB treated MIF demonstrated the strongest binding signal. To elucidate which cysteine residue of the MIF molecule (C57, C60 or C81) was modified by the sulfhydryl reactive compounds and therefore contributed to the structural change required for anti-oxMIF antibody binding, we first applied Matrix-assisted laser desorption/ionization (MALDI-TOF) mass spectrometry to determine the number of cysteine residues modified. The results demonstrate that L-cystine and DTNB treatment resulted in an increased molecular weight of +119 Da and +197 Da, respectively. L-cystine treated MIF (Cys-MIF) showed a peak at m/z 12342, corresponding to non-modified MIF and a peak at m/z 12461 (+119 Da) corresponding to a single cysteine adduct. DTNB treated-MIF (DTNB-MIF) also showed two main peaks, at m/z 12346 corresponding to non-modified MIF and m/z 12547 (+201 Da) corresponding to a single NTB moiety (Figure 2B). Additional peaks at m/z 12557 and 12561 of Cys-MIF and unmodified MIF, respectively, correspond to sinapinic acid matrix adducts generated during sample analysis. These data verified that only one cysteine of the MIF molecule is covalently modified by Lcystine or DTNB treatment. According to the MALDI-TOF peak areas DTNB treatment led to a conversion of approximately 50% of redMIF to the oxMIF surrogate DTNB-MIF. In contrast, Lcystine treatment only led to a conversion of approximately 10% of redMIF to the oxMIF surrogate Cys-MIF (Figure 2B).


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Subsequently tryptic digests of MIF, DTNB-MIF and Cys-MIF were subjected to peptide mass fingerprinting. In both preparations only the peptide consisting of the amino acids L78LCGLLAER87 (988 Da; non-modified MIF; Figure 2C) was modified either by cysteine (1107 Da; Figure 2D) or NTB (1185 Da; Figure 2E). The C57ALC60-motif spanning peptides did not harbor NTB or cysteine-adducts (data not shown). ELISA and mass spectrometry finally confirmed that only C81 is modified by both sulfhydryl reactive compounds. This leads to the assumption that C81 serves as a molecular redox switch playing a key role for inducing the structural rearrangement from redMIF to oxMIF.


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Figure 2 Binding properties (ELISA) and mass spectrometry characterization of oxMIF surrogates. (A) Binding of E.coli-derived recombinant human MIF (50 ng/ml) in its reduced state or MIF modified with different thiol-reactive reagents to immobilized oxMIF-specific antibodies BaxB01 (black bars) and BaxM159 (grey bars), and to matched human isotype IgG1 (Ctr IgG, white bars). (B) MALDI-TOF analysis of MIF, Cys-MIF (green line) DTNB-MIF (red line) and unmodified MIF (blue line). (C-E) Clipped spectra of a Peptide Mass Fingerprint of tryptic digest of unmodified MIF (blue line), Cys-MIF (green line) and DTNB-MIF (red line), for the peptide 78-86 harboring a cysteine (D) or NTB (E) adduct.

Substitution of MIF cysteines confirms that C81 is key for the structural rearrangement of redMIF to oxMIF. To confirm that C81 rather than the catalytic center cysteines C57 and C60 contribute to the structural change which is required for anti-oxMIF antibody binding, and to better characterize the mechanism that leads to the conversion of reduced MIF to oxMIF, we generated isosteric cysteine-to-serine mutants of MIF. The cysteine residues C57, C60, and C81 were substituted for serine and a streptavidin binding protein (SBP) tag was fused to the C-terminus of the protein with a flexible (GGGGS)2 linker. We assessed the binding of either non-modified or oxidized MIF(wt/mutant)-SBP fusion proteins to the antibodies BaxB01 and BaxM159 (Figure 3A, B). The ELISA using oxMIF specific antibody BaxM159 confirmed that modification of sulfhydryl groups with DTNB induced the formation of oxMIF which is recognized by the antibody. After conversion to oxMIF with DTNB, BaxM159 bound MIF(C57S)-SBP and MIF(C60S)-SBP to a degree that was comparable to its binding to DTNB treated MIF(wt)-SBP (Figure 3A). As expected, BaxM159 did not bind to non-treated MIF in its reduced state and lost binding after reduction of DTNB-MIF by DTT. However, when C81 was replaced by serine, treatment with DTNB did not enhance the binding of oxMIF-specific BaxM159 (Figure 3A). These data indicate, that an oxMIF structure can still be induced when C57 or C60 are replaced by serine, however, C81 is essential for effective conversion of redMIF to oxMIF. Using oxMIF specific antibody BaxB01 confirmed that oxMIF Seite 15 von 31 ACS Paragon Plus Environment

Biochemistry

can be induced by DTNB as the antibody did not recognize non-treated MIF nor MIF(wt)-SBP, but DTNB treated MIF species (Figure 3B). Again, substitution of C81 abolished the ability of MIF to form an oxMIF structure. However, no binding was observed when C57 or C60 were replaced by serine (Figure 3B), as these amino acids were part of the linear epitope of BaxB01 (Figure 1A-C).

OD

A

OD

B

C

OD

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3 Binding of MIF cysteine mutants to anti-oxMIF antibodies. (A-B) Binding of recombinant MIF or MIF(wt/mutant)-SBP(S) fusion proteins (50 ng/ml) in their unmodified form (grey bars), modified with DTNB (black bars) and reduced with DTT upon DTNB treatment (white bars) to immobilized oxMIF-specific antibodies BaxM159 (A) and BaxB01 (B). (C) Binding of MIF, Cys-MIF, MIF C81 mutants and MIF(A39E) mutant (50 ng/ml) to oxMIF-specific antibody BaxB01 (black bars) and to matched human isotype IgG (white bars).


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Biochemistry

The MIF(wt)-SBP and the mutants MIF(C57S)-SBP, MIF(C60S)-SBP and MIF(C81S)-SBP were furthermore treated with DTNB and a tryptic digest was analyzed by peptide mass fingerprinting. As expected, TNB adducts were found only in the peptide consisting of the amino acids L78LCGLLAER87 of MIF(wt)-SBP, MIF(C57S)-SBP and MIF(C60S)-SBP. However, no NTB adduct was detected in the MIF(C81S)-SBP after treatment with DTNB (data not shown). In summary, our ELISA and mass spectrometry data confirmed that only C81 is modified by sulfhydryl reactive compounds, leading to the assumption that C81 serves as a molecular redox switch playing a key role for inducing the structural rearrangement from reduced MIF to oxMIF. Substitution of C81 generates oxMIF surrogates. Chemical substitution converted only a fraction of redMIF to oxMIF, making subsequent functional and structural studies on this mixture difficult. In order to illuminate the structural importance of C81, we generated mutants where C81 was replaced by different amino acids, i.e. serine (C81S), glutamic acid (C81E) or tryptophan (C81W). All three mutants enabled binding of the oxMIF specific antibody BaxB01 and can be considered as oxMIF surrogates (Figure 3C). However, the ELISA signal correlated with the size of the amino acid that replaced C81 and the C81W mutant exhibited the strongest binding to oxMIF antibodies (Figure 3C). In addition, we generated mutants of alanine 39 which resides at the edge of the interacting adjacent beta sheets from two MIF monomers, which might help to enlighten whether or not an opening or disruption of the MIF trimer is crucial for anti-oxMIF antibody binding. Also the A39E mutant showed similar binding properties to BaxB01 compared to Cys-MIF (Figure 3C). We concluded that a significant structural change is taking place when redMIF is converted to oxMIF and C81 functions as a “switch” which reacts with a bulky moiety. Based on our result with the A39E


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mutant we furthermore postulated that an opening of the barrel structure and/or disruption of the MIF trimer occurs during conversion of redMIF to oxMIF. Characterization of MIF by circular dichroism (CD) studies confirms a structural switch by oxidation or mutation of C81. We determined the degree of the structural change in the tertiary and secondary structure of redMIF and C81 mutated oxMIF surrogates by CD studies in the Near- and Far-UV spectrum, respectively. Mutation of C81 resulted in a uniform oxMIF surrogate compared to chemical modification and we observed a characteristic shape of the spectra with distinct peaks in the wavelength region of aromatic amino acid residues (signals from 250-300 nm). High tension observed for all samples was comparable, confirming similar protein concentrations (data not shown). When oxMIF surrogates MIF(C81W), MIF(C81E), MIF(C81S) and the mutant MIF(A39E) were compared to redMIF, a strong amplitude reduction was observed in the NearUV CD spectrum (Figure 4A). This suggests a dramatic change in tertiary structure for these three samples as compared to the redMIF.


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Biochemistry

A

B

Figure 4 Near- and Far-UV CD spectra of MIF and MIF mutants. (A-B) Near-UV (A) and far UV (B) CD spectra of MIF, MIF(C81W), MIF (C81E), MIF(C81S) and MIF(A39E) at 1 mg/ml.

To estimate the overall change in the secondary structure of the different MIF isoforms and mutants, we analyzed the Far-UV CD spectrum and estimated the secondary structure by using the SDP48 reference database. All mutants show a change in the observed spectrum compared to redMIF, especially in the wavelength region between 190 nm and 210 nm (Figure 4B). The comparison of the secondary structure of MIF with its mutants demonstrates that the content of alpha helix and turn remained essentially constant for all MIF and MIF-mutant samples (Table 1). However, the oxMIF surrogate mutants showed lower beta sheet content and slightly higher random coil content. This structural change is most prominent for the MIF(C81W) mutant with a decrease of beta sheet content to 13% (Table 1). This relates to our previous findings which Seite 19 von 31 ACS Paragon Plus Environment

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indicated that the conversion of redMIF to oxMIF is most pronounced when C81 is loaded with a large moiety.

Sample

alpha helix [%]

beta sheet [%]

turn [%]

random [%]

RMSD

MIF

37.2

25.5

16.6

20.6

0.105

MIF(C81W)

38.9

13.5

18.4

29.2

0.126

MIF(C81E)

36.8

18.0

18.2

27.0

0.100

MIF(C81S)

37.0

16.8

17.9

28.2

0.110

36.7 17.6 17.1 28.6 0.129 MIF(A39E) Table 1 Secondary structure estimation of MIF and MIF mutants. Far-UV spectra were analyzed by using a CDPro and the CONTIN algorithm.

Furthermore, we conducted sedimentation velocity analytical ultracentrifugation (SV-AUC) studies to elucidate the oligomerization state of MIF and MIF mutants. However, the SV-AUC analyses revealed no major differences in the abundance of trimer or high molecular weight species (HMWS), irrespective of the mutation (Table 2). All samples exhibited virtually identical high trimer and low HMWS contents of 98.3-99.5% and 0.7-1.7%, respectively. The sedimentation coefficient of 3.0-3.1 S for the trimer was almost identical for all these samples, and also the frictional ratios of 1.19-1.26 did not differ significantly. Sample

Species Content [%] s [S] MW [kDa] f/f0 MIF Trimer 99.3 ± 0.3 3.0 ± 0 33 ± 0 MIF 1.23 ± 0.01 HMWS 0.7 ± 0.3 6.7-11.2 112-237 MIF Trimer 98.6 ± 0.1 3.0 ± 0 34 ± 0 MIF(C81W) 1.26 ± 0 HMWS 1.4 ± 0.1 6.8-9.6 117-195

MIF Trimer 98.7 ± 0.7 HMWS 1.3 ± 0.7 MIF Trimer 99.3 ± 0.2 MIF(C81S) HMWS 0.7 ± 0.2 MIF(C81E)

3.0 ± 0 6.8-10.8

34 ± 0 117-231

1.26 ± 0

2.9 ± 0 7.2-9.4

32 ± 0 125-188

1.25 ± 0.01

MIF Trimer 97.5 ± 0.5 3.0 ± 0 31 ± 1 1.19 ± 0.02 HMWS 2.5 ± 0.5 5.1-9.1 68-168 Table 2 Molecular weight estimation from various MIF species and mutants by SV-AUC. Sedimentation profiles were analyzed with Sedfit. MIF(A39E)


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Biochemistry

In summary, oxMIF surrogates remained in its homo-trimeric conformation, but the reduced beta sheet and higher random coil content suggests an opening/loosening of the tight MIF trimer which enables accessibility and binding of anti-oxMIF antibodies. Structural evidence for TPOR activity regulation accompanying redMIF to oxMIF transition. To decipher the mechanism of recognition of oxMIF by BaxB01 we determined the crystal structure of the BaxB01-Fab domain alone (“apo”) (Figure 5A, Table 3) and in complex with the extended linear epitope EPC57ALC60S (Figure 5B, Table 3). The overall structure of BaxB01Fab adopted a typical immunoglobulin-like fold and was structurally conserved in the apo and the complex structure, with only minor structural adaptions in its paratope. The EPC57ALC60S epitope adopted a strongly curved conformation in the complex with BaxB01-Fab, in stark contrast to the extended conformation of this peptide in the crystal structure of homotrimeric redMIF (Figure 5B, C). To unambiguously assign the binding mode of the EPCALCS peptide, we used seleno-cysteine variants of the epitope peptide, where the sulfur-cysteine is replaced by seleno-cysteine. The seleno-cysteine sC60 was exploited for its anomalous scattering signal, which unambiguously positioned the selenium (Figure 5C). The electrostatic surface potential of the Fab paratope demonstrates the overall hydrophobic character with a slightly positively charged patch, consistent with the negative charge of the epitope’s E55 (Figure 5C). Importantly, the BaxB01-bound epitope brought the side chains of C57 and C60 into proximity, similar as seen for the CXXC motif in thioredoxin-like protein disulfide isomerases25. The complex structure thus appears to trap the oxidoreductase-active site in an active state, where disulfide shuffling is enabled. In contrast, the reduced state keeps the active site in an cryptic, zymogen-


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like state, where the cysteines are in an extended conformation, unable to undergo disulfide shuffling (Figure 5D).

Figure 5 Crystal structure of oxMIF specific Fab fragment BaxB01 alone and in complex with the MIF epitope EPCALCS. (A) Cartoon representation of the Fab fragment of Baxb01, with heavy and light chain indicated. The CDR loops are situated at the top and labeled with H1-3 and L1-3 for heavy and light chain respectively. (B) Electrostatic surface rendering of BaxB01 Fab in complex with EPCALCS peptide show an overall hydrophobic binding site. (C) The experimental electron density (green) overlaid with the anomalous density (cyan) unambiguously determine the position of the selenium atom of Cys60 (Seγ). (D) Model of the conformational maturation of MIF upon binding to BaxB01.


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Biochemistry

Data collection

BaxB01-Fab

Space group

P41212

Unit cell (axes in Å, angles in °)

a = b = 102.70, c = 188.69, α = β = γ = 90.0

Wavelength in Å

0.96770

Resolution in Å (highest shell)

72.62 - 2.60 (2.74 - 2.60)

Unique reflections/multiplicity

31799 (4522)/17.4 (16.4)

Completeness

0.997 (0.993)

mean I/sigma(I)

10.6 (1.5)

Rmerge/Rpim (precision indicating)

0.204 (1.960) /0.049 (0.480)

CC1/2 (correlation half data sets)

0.995 (0.630)

Refinement

BaxB01-Fab

Resolution range in Å (highest shell)

69.50 - 2.60 (2.68 - 2.60)

Reflections working set/test set for Rfree

31694 (2812) /1537 (124)

Rwork/Rfree

21.4 (30.9) /26.3 (39.8)

Protein residues

878 (2 Fab per asu)

Protein atoms [average B-factor]

6646 [64.47]

rmsd bonds/angles

0.004/0.675

Ramachandran plot (favored/allowed/outliers) 95.3%/4.7%/ Data collection

BaxB01-Fab EPCALSecS

Space group

C2

Unit cell (axes in Å, angles in °)

a = 307.81 b = 218.75, c = 222.92, α = γ = 90.0, β = 108.16

Wavelength in Å

0.97901

Resolution in Å (highest shell)

211.82 - 3.80 (4.01 - 3.80)

Unique reflections/multiplicity

137440 (19992)/3.9 (3.8)

Completeness

0.997 (0.995)

mean I/sigma(I)

10.0 (1.5)

Rmerge/Rpim (precision indicating)

0.088 (1.063) /0.051 (0.637)

CC1/2 (correlation half data sets)

0.999 (0.756)

Refinement

BaxB01-Fab EPCALSecS

Resolution range in Å (highest shell)

105.91 – 3.80 (3.94 - 3.80)

Reflections working set/test set for Rfree

136603 (12837) /6849 (659)

Rwork/Rfree

26.4 (33.0) /34.6 (43.4)

Protein residues

10536 (24 Fab per asu)

Protein atoms [average B-factor]

79776 [175.9]

rmsd bonds/angles

0.003/0.706

Ramachandran plot (favored/allowed/outliers) 89.1%/10.2%/0.7% Table 3 Data collection and refinement statistics for BaxB01-Fab and BaxB01-Fab in complex with the Selenocysteine (Sec) containing peptide EPCALSecS. Data of the highest resolution shells are shown in parentheses.


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DISCUSSION MIF occurs in two immunologically distinct conformational isoforms18, 19, redMIF and oxMIF, which reversibly can interconvert in vitro dependent upon the local redox potential. Monoclonal antibodies BaxB01 and BaxM159, which were shown to be protective in animal models of inflammation and cancer, were described to exclusively bind to oxMIF and not to recognize redMIF18, 19, 20, 21. The constitutive expression and wide tissue distribution of MIF in diseased and healthy subjects is one of the most unusual properties of a proinflammatory cytokine and became further explainable by the oxMIF/redMIF concept: OxMIF was found to be the disease related isoform that is predominantly expressed in patients with inflammatory diseases and cancer, whereas redMIF represents the ubiquitous storage form of MIF18, 19. The conversion of redMIF to the disease-related protein isoform was mimicked in vitro by mild oxidation18. We set out to investigate a potential involvement of the three free cysteines (C57, C60 and C81) in the transition to oxMIF. Our data demonstrated that sulfhydryl reactive compounds like L-cystine, MMTS or DTNB, form covalent disulfide bonds with C81 only, whereas C57 or C60 remain as free thiols. Thus, only C81 serves as a ”key switch” by which redMIF can be converted to oxMIF via a post-translational modification or protein-protein interaction. C81 is surrounded by mostly hydrophobic residues and exhibits enhanced accessibility compared to C57 and C6017. The crystal structure of MIF was solved in the 1990s22, 23 and according to this structure the epitopes of oxMIF specific antibodies BaxB01 and BaxM159 are buried in the core of the MIF trimer which is consistent with the finding that these antibodies do not recognize MIF in its reduced state. A significant structural rearrangement is required during the switch from redMIF Seite 24 von 31 ACS Paragon Plus Environment

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Biochemistry

to oxMIF to allow the antibodies to access their epitopes. According to our data this rearrangement is induced by adducts to C81, leading to a lower beta sheet content and higher random coil content compared to non-modified redMIF. Despite a structural rearrangement, the trimeric quaternary structure is maintained. Our studies using C81 mutants of MIF showed that the conversion is more pronounced when the space of C81 is filled with a bulky moiety. The structural rearrangement of MIF during redMIF to oxMIF conversion is also demonstrated by the crystal structure of the extended epitope of antibody BaxB01 (EPCALCS) which was kept in its oxMIF-like conformation by complexation with the BaxB01-Fab fragment. The crystal structure revealed a curved conformation of the EPCALCS epitope which brings the cysteine thiols in close proximity, potentially enabling redox reactions. This oxMIF like structure of the EPCALCS peptide could represent a physiologically relevant, i.e. enzymatically active state, where the catalytic cysteines C57 and C60 are in proximity to undergo disulfide bridge closure and opening. This structure thus suggests functional analogy of oxMIF to thioredoxin and other CXXC oxidoreductases25. In contrast, in the trimeric crystal structure of redMIF, the thiols of the catalytic cysteine residues point towards opposite sides of the β sheet in which the peptide EPCALCS is incorporated. Consequently, this structure could represent a zymogen form. Taken together, our data support the hypothesis (i) that redMIF represents a ubiquitous storage form of MIF where the molecule is kept in a zymogen form. (ii) Upon disease development, modification of C81 mediates the conversion of redMIF to oxMIF which exerts activity relevant for pathophysiological processes. (iii) OxMIF specific antibodies BaxB01 or BaxM159 block functional properties oxMIF as their epitopes are in close proximity to the CXXC catalytic center interfering with disulfide shuffling.


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

However, it is currently not known which proteins and factors are involved in redMIF to oxMIF conversion in vivo and how this process is regulated. Previous reports support the idea that MIFs activity is tightly regulated by redox mechanisms26. Many intracellular proteins have been identified as MIF binding partners with some emphasizing the importance of C81 (reviewed in 27

). In contrast to the CXXC motif cysteines C57 and C60, the role of C81 has been barely

investigated so far27. It will be important to further elucidate redox-dependent functions of C81 in inflammatory processes (i.e. modulation of oxidoreductase activity via C81, MIF protein-protein interactions and MIF cellular expression patterns). In addition, it is also not clear how oxMIF mediates cellular signal transduction and how this mechanism differentiates oxMIF from redMIF. Different MIF receptors have been reported28-30, but their interactions with different MIFisoforms have, to our knowledge, not been described so far. Thus, even more than 50 years after MIF has been described for the first time31, many question remain to be elucidated. Nevertheless, the involvement of oxMIF in inflammatory diseases and cancer and the beneficial effect of oxMIF specific antibodies in vitro and in vivo make it a promising target for therapeutic intervention.


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Biochemistry

AUTHOR INFORMATION Corresponding authors Michael Thiele, Baxalta Innovations GmbH (part of Shire), Uferstrasse 15, Orth an der Donau, 2304, Austria, [email protected], Phone: +43 1 20100 244 4908 Friedrich Scheiflinger, Baxalta Innovations GmbH (part of Shire), Donau-City-St. 7, 1220 Vienna, 1220, Austria, [email protected], Phone: +43 1 20100 2473410 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources Research was solely funded by Baxter, Baxalta and Shire


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ABBREVIATIONS Akt, protein kinase B; Bis/Tris, bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane; BSA, bovine serum albumin; CD, circular dichroism; DMSO, dimethyl sulfoxide; DTNB, 5,5'-dithiobis-2-nitrobenzoic acid; DTT, dithiothreitol; E.coli, Escherichia coli; EDTA, ethylenediaminetetraacetic acid; ELISA, enzyme-linked immunosorbent assay; EMCA, nepsilon-maleimidocaproic acid; ERK1/2, extracellular signal-regulated kinase 1/2; FG, fish gelatin; GSSG, oxidized glutathione; IL-8, interleukin 8; MMTS, methyl methanethiosulfonate; p53, tumor suppressor protein p53; PBS, phosphate-buffered saline; PEG, polyethylene glycol; PI3K, phosphoinositide 3-kinase; TBST, tris-buffered saline Tween 20; TPOR, thiol-protein oxidoreductase; Tris, tris(hydroxymethyl)aminomethane


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Biochemistry

REFERENCES [1] Bacher, M., Meinhardt, A., Lan, H. Y., Mu, W., Metz, C. N., Chesney, J. A., Calandra, T., Gemsa, D., Donnelly, T., Atkins, R. C., and Bucala, R. (1997) Migration inhibitory factor expression in experimentally induced endotoxemia, Am J Pathol 150, 235-246. [2] Flieger, O., Engling, A., Bucala, R., Lue, H., Nickel, W., and Bernhagen, J. (2003) Regulated secretion of macrophage migration inhibitory factor is mediated by a non-classical pathway involving an ABC transporter, FEBS Lett 551, 78-86. [3] Merk, M., Baugh, J., Zierow, S., Leng, L., Pal, U., Lee, S. J., Ebert, A. D., Mizue, Y., Trent, J. O., Mitchell, R., Nickel, W., Kavathas, P. B., Bernhagen, J., and Bucala, R. (2009) The Golgi-associated protein p115 mediates the secretion of macrophage migration inhibitory factor, J Immunol 182, 6896-6906. [4] Emonts, M., Sweep, F. C., Grebenchtchikov, N., Geurts-Moespot, A., Knaup, M., Chanson, A. L., Erard, V., Renner, P., Hermans, P. W., Hazelzet, J. A., and Calandra, T. (2007) Association between high levels of blood macrophage migration inhibitory factor, inappropriate adrenal response, and early death in patients with severe sepsis, Clin Infect Dis 44, 1321-1328. [5] Regis, E. G., Barreto-de-Souza, V., Morgado, M. G., Bozza, M. T., Leng, L., Bucala, R., and Bou-Habib, D. C. (2010) Elevated levels of macrophage migration inhibitory factor (MIF) in the plasma of HIV-1-infected patients and in HIV-1-infected cell cultures: a relevant role on viral replication, Virology 399, 31-38. [6] Mitchell, R. A., and Bucala, R. (2000) Tumor growth-promoting properties of macrophage migration inhibitory factor (MIF), Semin Cancer Biol 10, 359-366. [7] Lue, H., Kleemann, R., Calandra, T., Roger, T., and Bernhagen, J. (2002) Macrophage migration inhibitory factor (MIF): mechanisms of action and role in disease, Microbes Infect 4, 449-460. [8] Lue, H., Thiele, M., Franz, J., Dahl, E., Speckgens, S., Leng, L., Fingerle-Rowson, G., Bucala, R., Luscher, B., and Bernhagen, J. (2007) Macrophage migration inhibitory factor (MIF) promotes cell survival by activation of the Akt pathway and role for CSN5/JAB1 in the control of autocrine MIF activity, Oncogene 26, 5046-5059. [9] Kim, H. R., Park, M. K., Cho, M. L., Yoon, C. H., Lee, S. H., Park, S. H., Leng, L., Bucala, R., Kang, I., Choe, J., and Kim, H. Y. (2007) Macrophage migration inhibitory factor upregulates angiogenic factors and correlates with clinical measures in rheumatoid arthritis, J Rheumatol 34, 927-936. [10] Bondza, P. K., Metz, C. N., and Akoum, A. (2008) Macrophage migration inhibitory factor up-regulates alpha(v)beta(3) integrin and vascular endothelial growth factor expression in endometrial adenocarcinoma cell line Ishikawa, J Reprod Immunol 77, 142-151. [11] Rosengren, E., Bucala, R., Aman, P., Jacobsson, L., Odh, G., Metz, C. N., and Rorsman, H. (1996) The immunoregulatory mediator macrophage migration inhibitory factor (MIF) catalyzes a tautomerization reaction, Mol Med 2, 143-149. [12] Rosengren, E., Aman, P., Thelin, S., Hansson, C., Ahlfors, S., Bjork, P., Jacobsson, L., and Rorsman, H. (1997) The macrophage migration inhibitory factor MIF is a phenylpyruvate tautomerase, FEBS Lett 417, 85-88. [13] Kleemann, R., Mischke, R., Kapurniotu, A., Brunner, H., and Bernhagen, J. (1998) Specific reduction of insulin disulfides by macrophage migration inhibitory factor (MIF) with glutathione and dihydrolipoamide: potential role in cellular redox processes, FEBS Lett 430, 191-196. [14] Hermanowski-Vosatka, A., Mundt, S. S., Ayala, J. M., Goyal, S., Hanlon, W. A., Czerwinski, R. M., Wright, S. D., and Whitman, C. P. (1999) Enzymatically inactive macrophage migration inhibitory factor inhibits monocyte chemotaxis and random migration, Biochemistry 38, 12841-12849.


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[15] Swope, M., Sun, H. W., Blake, P. R., and Lolis, E. (1998) Direct link between cytokine activity and a catalytic site for macrophage migration inhibitory factor, EMBO J 17, 35343541. [16] Fingerle-Rowson, G., Kaleswarapu, D. R., Schlander, C., Kabgani, N., Brocks, T., Reinart, N., Busch, R., Schutz, A., Lue, H., Du, X., Liu, A., Xiong, H., Chen, Y., Nemajerova, A., Hallek, M., Bernhagen, J., Leng, L., and Bucala, R. (2009) A tautomerase-null macrophage migration-inhibitory factor (MIF) gene knock-in mouse model reveals that protein interactions and not enzymatic activity mediate MIF-dependent growth regulation, Mol Cell Biol 29, 1922-1932. [17] Kleemann, R., Kapurniotu, A., Mischke, R., Held, J., and Bernhagen, J. (1999) Characterization of catalytic centre mutants of macrophage migration inhibitory factor (MIF) and comparison to Cys81Ser MIF, Eur J Biochem 261, 753-766. [18] Thiele, M., Kerschbaumer, R. J., Tam, F. W., Volkel, D., Douillard, P., Schinagl, A., Kuhnel, H., Smith, J., McDaid, J. P., Bhangal, G., Yu, M. C., Pusey, C. D., Cook, H. T., Kovarik, J., Magelky, E., Bhan, A., Rieger, M., Mudde, G. C., Ehrlich, H., Jilma, B., Tilg, H., Moschen, A., Terhorst, C., and Scheiflinger, F. (2015) Selective Targeting of a DiseaseRelated Conformational Isoform of Macrophage Migration Inhibitory Factor Ameliorates Inflammatory Conditions, J Immunol. [19] Schinagl, A., Thiele, M., Douillard, P., Volkel, D., Kenner, L., Kazemi, Z., Freissmuth, M., Scheiflinger, F., and Kerschbaumer, R. J. (2016) Oxidized macrophage migration inhibitory factor is a potential new tissue marker and drug target in cancer, Oncotarget 7, 73486-73496. [20] Kerschbaumer, R. J., Rieger, M., Volkel, D., Le Roy, D., Roger, T., Garbaraviciene, J., Boehncke, W. H., Mullberg, J., Hoet, R. M., Wood, C. R., Antoine, G., Thiele, M., SavidisDacho, H., Dockal, M., Ehrlich, H., Calandra, T., and Scheiflinger, F. (2012) Neutralization of macrophage migration inhibitory factor (MIF) by fully human antibodies correlates with their specificity for the beta-sheet structure of MIF, J Biol Chem 287, 7446-7455. [21] Hussain, F., Freissmuth, M., Volkel, D., Thiele, M., Douillard, P., Antoine, G., Thurner, P., Ehrlich, H., Schwarz, H. P., Scheiflinger, F., and Kerschbaumer, R. J. (2013) Human anti-macrophage migration inhibitory factor antibodies inhibit growth of human prostate cancer cells in vitro and in vivo, Mol Cancer Ther 12, 1223-1234. [22] Sun, H. W., Bernhagen, J., Bucala, R., and Lolis, E. (1996) Crystal structure at 2.6-A resolution of human macrophage migration inhibitory factor, Proc Natl Acad Sci U S A 93, 5191-5196. [23] Suzuki, M., Sugimoto, H., Nakagawa, A., Tanaka, I., Nishihira, J., and Sakai, M. (1996) Crystal structure of the macrophage migration inhibitory factor from rat liver, Nat Struct Biol 3, 259-266. [24] Taylor, A. B., Johnson, W. H., Jr., Czerwinski, R. M., Li, H. S., Hackert, M. L., and Whitman, C. P. (1999) Crystal structure of macrophage migration inhibitory factor complexed with (E)-2-fluoro-p-hydroxycinnamate at 1.8 A resolution: implications for enzymatic catalysis and inhibition, Biochemistry 38, 7444-7452. [25] Quan, S., Schneider, I., Pan, J., Von Hacht, A., and Bardwell, J. C. (2007) The CXXC motif is more than a redox rheostat, J Biol Chem 282, 28823-28833. [26] Thiele, M., Bernhagen, J. (2005) Link between macrophage migration inhibitory factor and cellular redox regulation, Antioxid Redox Signal 7, 1234-1248. [27] Meinhardt, J. K. a. A. (2012) The MIF Handbook, Vol. 1, World Scientific Publishing Co. Pte. Ltd., Singapore. [28] Leng, L., Metz, C. N., Fang, Y., Xu, J., Donnelly, S., Baugh, J., Delohery, T., Chen, Y., Mitchell, R. A., and Bucala, R. (2003) MIF signal transduction initiated by binding to CD74, J Exp Med 197, 1467-1476.


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Biochemistry

[29] Shi, X., Leng, L., Wang, T., Wang, W., Du, X., Li, J., McDonald, C., Chen, Z., Murphy, J. W., Lolis, E., Noble, P., Knudson, W., and Bucala, R. (2006) CD44 is the signaling component of the macrophage migration inhibitory factor-CD74 receptor complex, Immunity 25, 595-606. [30] Bernhagen, J., Krohn, R., Lue, H., Gregory, J. L., Zernecke, A., Koenen, R. R., Dewor, M., Georgiev, I., Schober, A., Leng, L., Kooistra, T., Fingerle-Rowson, G., Ghezzi, P., Kleemann, R., McColl, S. R., Bucala, R., Hickey, M. J., and Weber, C. (2007) MIF is a noncognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment, Nat Med 13, 587-596. [31] Bloom, B. R., and Bennett, B. (1966) Mechanism of a reaction in vitro associated with delayed-type hypersensitivity, Science 153, 80-82. [32] Battye, T. G., Kontogiannis, L., Johnson, O., Powell, H. R., and Leslie, A. G. (2011) iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM, Acta Crystallogr D Biol Crystallogr 67, 271-281. [33] Evans, P. (2006) Scaling and assessment of data quality, Acta Crystallogr D Biol Crystallogr 62, 72-82. [34] Winn, M. D., Ballard, C. C., Cowtan, K. D., Dodson, E. J., Emsley, P., Evans, P. R., Keegan, R. M., Krissinel, E. B., Leslie, A. G., McCoy, A., McNicholas, S. J., Murshudov, G. N., Pannu, N. S., Potterton, E. A., Powell, H. R., Read, R. J., Vagin, A., and Wilson, K. S. (2011) Overview of the CCP4 suite and current developments, Acta Crystallogr D Biol Crystallogr 67, 235-242. [35] McCoy, A. J. (2007) Solving structures of protein complexes by molecular replacement with Phaser, Acta Crystallogr D Biol Crystallogr 63, 32-41. [36] Vaguine, A. A., Richelle, J., and Wodak, S. J. (1999) SFCHECK: a unified set of procedures for evaluating the quality of macromolecular structure-factor data and their agreement with the atomic model, Acta Crystallogr D Biol Crystallogr 55, 191-205. [37] Emsley, P., Lohkamp, B., Scott, W. G., and Cowtan, K. (2010) Features and development of Coot, Acta Crystallogr D Biol Crystallogr 66, 486-501. [38] Murshudov, G. N., Skubak, P., Lebedev, A. A., Pannu, N. S., Steiner, R. A., Nicholls, R. A., Winn, M. D., Long, F., and Vagin, A. A. (2011) REFMAC5 for the refinement of macromolecular crystal structures, Acta Crystallogr D Biol Crystallogr 67, 355-367. [39] Afonine, P. V., Grosse-Kunstleve, R. W., Echols, N., Headd, J. J., Moriarty, N. W., Mustyakimov, M., Terwilliger, T. C., Urzhumtsev, A., Zwart, P. H., and Adams, P. D. (2012) Towards automated crystallographic structure refinement with phenix.refine, Acta Crystallogr D Biol Crystallogr 68, 352-367.


LRISPDRVYINYYDM LRISSDRVYINYYDM LRISPSRVYINYYDM LRISPDSVYINYYDM LRISPDRSYINYYDM LRISPDRVSINYYDM LRISPDRVYSNYYDM LRISPDRVYISYYDM LRISPDRVYINSYDM LRISPDRVYINYSDM LRISPDRVYINYYSM

FGGSEPCALCSLHSI FGGSSPCALCSLHSI FGGSESCALCSLHSI FGGSEPSALCSLHSI FGGSEPCSLCSLHSI FGGSEPCASCSLHSI FGGSEPCALSSLHSI FGGSEPCALCSLHSI FGGSEPCALCSSHSI

3000 PMFIVNTNVPRASVP MFIVNTNVPRASVPD FIVNTNVPRASVPDG IVNTNVPRASVPDGF VNTNVPRASVPDGFL NTNVPRASVPDGFLS TNVPRASVPDGFLSE NVPRASVPDGFLSEL VPRASVPDGFLSELT PRASVPDGFLSELTQ RASVPDGFLSELTQQ ASVPDGFLSELTQQL SVPDGFLSELTQQLA VPDGFLSELTQQLAQ PDGFLSELTQQLAQA DGFLSELTQQLAQAT GFLSELTQQLAQATG FLSELTQQLAQATGK LSELTQQLAQATGKP SELTQQLAQATGKPP ELTQQLAQATGKPPQ LTQQLAQATGKPPQY TQQLAQATGKPPQYI QQLAQATGKPPQYIA QLAQATGKPPQYIAV LAQATGKPPQYIAVH AQATGKPPQYIAVHV QATGKPPQYIAVHVV ATGKPPQYIAVHVVP TGKPPQYIAVHVVPD GKPPQYIAVHVVPDQ KPPQYIAVHVVPDQL PPQYIAVHVVPDQLM PQYIAVHVVPDQLMA QYIAVHVVPDQLMAF YIAVHVVPDQLMAFG IAVHVVPDQLMAFGG AVHVVPDQLMAFGGS VHVVPDQLMAFGGSS HVVPDQLMAFGGSSE VVPDQLMAFGGSSEP VPDQLMAFGGSSEPC PDQLMAFGGSSEPCA DQLMAFGGSSEPCAL QLMAFGGSSEPCALC LMAFGGSSEPCALCS MAFGGSSEPCALCSL AFGGSSEPCALCSLH FGGSSEPCALCSLHS GGSSEPCALCSLHSI GSSEPCALCSLHSIG SSEPCALCSLHSIGK SEPCALCSLHSIGKI EPCALCSLHSIGKIG PCALCSLHSIGKIGG CALCSLHSIGKIGGA ALCSLHSIGKIGGAQ LCSLHSIGKIGGAQN CSLHSIGKIGGAQNR SLHSIGKIGGAQNRS LHSIGKIGGAQNRSY HSIGKIGGAQNRSYS SIGKIGGAQNRSYSK IGKIGGAQNRSYSKL GKIGGAQNRSYSKLL KIGGAQNRSYSKLLC IGGAQNRSYSKLLCG GGAQNRSYSKLLCGL GAQNRSYSKLLCGLL AQNRSYSKLLCGLLA QNRSYSKLLCGLLAE NRSYSKLLCGLLAER RSYSKLLCGLLAERL SYSKLLCGLLAERLR YSKLLCGLLAERLRI SKLLCGLLAERLRIS KLLCGLLAERLRISP LLCGLLAERLRISPD LCGLLAERLRISPDR CGLLAERLRISPDRV GLLAERLRISPDRVY LLAERLRISPDRVYI LAERLRISPDRVYIN AERLRISPDRVYINY ERLRISPDRVYINYY RLRISPDRVYINYYD LRISPDRVYINYYDM RISPDRVYINYYDMN ISPDRVYINYYDMNA SPDRVYINYYDMNAA PDRVYINYYDMNAAN DRVYINYYDMNAANV RVYINYYDMNAANVG VYINYYDMNAANVGW YINYYDMNAANVGWN INYYDMNAANVGWNN NYYDMNAANVGWNNS YYDMNAANVGWNNST YDMNAANVGWNNSTF DMNAANVGWNNSTFA

OD

1 2A 3 4 5 6 7 8 9 10 11 12 13 14 15 16 C 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

OD

Biochemistry

B 3000

2000

D E

1000

0

ACS Paragon Plus Environment

Page 32 of 37

P56CALC60 D93RVYINYY100

2000

1000

0

F

4 .0

Biochemistry

B

B axB 01

12346 12342 12346

B axM 159

12547

Is o ty p e

Rel. Intensity (%)

3 .0

2 .0

12461

1 .0

D

DTNB

M M TS

L - C y s t in

C

EM C A

0 .0 u n tr e a te d

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

A

OD

Page 33 of 37

E

E


Biochemistry

A

3 .0

u n tre a te d D TNB D T N B & re d u c e d

OD

2 .0

1 .0

0 .0

MI

B

F

F( MI

wt

)-S

5 F (C

7S

)-S

MI MI u n tre a te d D TNB D T N B & re d u c e d

3 .0

6 F (C

0S

)-S MI

8 F (C

1S

)-S

OD

2 .0

1 .0

0 .0

MI

C

F

1 .5

F( MI

wt

)-S

M

C5 IF (

7S

)-S M

C6 IF (

0S

)-S M

C8 IF (

1S

)-S

B axB 01 Is o ty p e

1 .0

OD

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

Page 34 of 37

0 .5

0 .0

MI

F

Cy

) ) ) IF W) 9E 1S 1E sM 81 A3 C8 C8 ( C ( ( ( F F F I F I I ACS M M Paragon M Plus Environment MI

Page 35 of 37



m r

[d e g c m

2

-1

dm ol ]

A 320

MIF

280

M IF (C 8 1 W ) M IF (C 8 1 E )

240

M IF (C 8 1 S )

200

M IF (A 3 9 E )

160 120 80 40 0 240

260

280

300

320

w a v e le n g th (n m )

B

MIF M IF (C 8 1 W )

-1

dm ol ]

30000

M IF (C 8 1 E )

20000

M IF (C 8 1 S ) M IF (A 3 9 E )

m r

[d e g c m

2

10000 0 -1 0 0 0 0



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

Biochemistry

-2 0 0 0 0 200

220

w a v e le n g th (n m )


240

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

A

B

C

D


Page 36 of 37

Page 37 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

82x44mm (300 x 300 DPI)


Role of the Cysteine 81 Residue of Macrophage Migration Inhibitory

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