Purpose(s)
Determine the role of MIF N-terminus in activation of CD74.
Probe the potential dynamic pathway that connects the N-terminus with the MIF surface responsible for activation of CD74.
Analyze the factors that distinguish MIF variants serving as CD74 antagonists or agonists.
Method(s)
Mutagenesis, Protein Expression, and Purification. WT MIF and MIF variants were grown at 37C to an OD600 of 0.6-0.8 and induced with 1mM IPTG for 4 hours at 37C. MIF proteins were purified using ion exchange (Q- Sepharose) and size exclusion (16/60 Superdex 75) chromatography. Protein concentration was determined with the Pierce™ BCA protein assay kit (Thermo Fisher Scientific).
Circular Dichroism (CD) Spectroscopy. Secondary structure scans and thermal denaturation experiments were carried out in a 1 mm quartz cuvette using a JASCO J-810 spectropolarimeter.
MD Simulations. 200ns molecular dynamics simulations were performed for WTMIF and MIF variants. The systems’ waters, side chains, and then entirety were then minimized (20ps), heated to 300K (120ps), and equilibrated (1ns). Generalized cross correlation data for -carbon atoms and RMSF values were produced with GROMACS. RMSD data were obtained using SUPERPOSE and all models were analyzed in PyMOL.
NMR experiments. 1H-15N TROSY HSQCs and spin relaxation experiments were carried out on a Bruker Avance NEO 600 MHz spectrometer at 30°C. The 1H and 15N carrier frequencies were set to water resonance and 120 ppm, respectively. All NMR spectra were processed with NMRpipe and analyzed in Sparky. Relaxation experiments were carried out in a temperature-compensated interleaved manner, processed with in-house scripts, and analyzed in GraphPad Prism 8.0 (GraphPad Software).
Result(s)
Figure 1. The MIF variants used in this study. The structure of monomeric WT MIF (PDB entry: 3DJH) is shown in black. The beta-strands are labeled and the catalytically active N-terminus amino acid, Pro1, is shown as sticks. Although the biological assembly of MIF is homotrimeric, we illustrate only one monomer for simplicity. The N-terminal sites of MIF variants P1M (yellow), P1G (blue), M2A (purple), and deltaP1 (red) are shown at the four corners and obtained from the PDB entries 4PKZ, 1P1G, 4XX7, and 4XX8 for P1M, P1G, M2A, and deltaP1, respectively. Notably, the N-terminus residue of deltaP1, Met2, adopts two conformations.
Figure 2. 1H-15N HSQC NMR spectral overlays of P1G (blue), P1M (yellow), deltaP1 (red), and M2A (purple) MIF with WT MIF (black).
Figure 3. Protein folding and stability analysis for WT MIF and the MIF variants. A. Folding profiles were monitored between 260nm and 195nm, at 25C. B. Thermal stability curves for WT MIF and MIF variants were determined at 218nm. C. CD spectroscopy shows a difference between the WT and DP1 that can be attributed to the loss of the rigid Pro1. Due to the increased flexibility of the N-terminus, Met2 can no longer participate in the b-sheet.
Figure 4. Global and local fluctuations of MIF variants as determined by 200ns MD simulations.
Figure 5. MIF variants serving as CD74 antagonists or agonist. N-terminus changes result in negative or positive cooperativity, as determined by MD simulations. In vivo CD74 activation assays, correlate negative and positive cooperativity with antagonist and agonistic effect, respectively.
Figure 6. Relaxation parameters of MIF variants vs. WT. A. R1R2 relaxation rates compare dynamics of WT MIF and the MIF variants. P1M and P1G both see broadening effects; this suggests that the disturbance of Pro1 results in dynamic changes to neighboring residues. B. This is also present in the 1H-[15N] NOE data. Both pieces of data support, that displacing Pro1 results in dynamic changes in residues 28-36, 44-49, and 60-68. Most of these are found on the beta2 and beta4 strands which aligns with the data from the MD simulations.
Conclusion(s)
The N-terminus regulates b-sheet flexibility and transmits dynamic signals to the surface through the b2 and b4 strands.
MIF variants serving as CD74 agonists increase the overall correlation of key amino acids with the MIF trimer, while MIF variants serving as CD74 antagonists decrease it.