Molecular Dynamics Simulation Of The Interaction Between Macrophage Migration Inhibitory Factor And Small Molecules

Macrophage migration inhibitory factor (MIF) is a pivotal regulator of innate immunity and plays an important role in the host antimicrobial alarm system and stress response that promotes the pro-inflammatory functions of immune cells. It has been implicated in the pathogenesis of sepsis, tumors, inflammatory and autoimmune diseases. MIF can also act as a phenylpyruvate tautomerase, D-dopachrome tautomerase and thiol-protein oxidoreductase. Since the accumulated acknowledgement for the special biological function and its role in many diseases, MIF has now become a research hotspot.The current research related to MIF is mainly limited to experiments at the cell level, while little research is performed for further study of the structure of MIF, enzymatic mechanism and interactions with inhibitors at atomic level. The detailed binding mode of MIF with different types of inhibitors, the bioactivity difference within certain a series of inhibitor analogues and the effects on the binding modes brought about by the mutation of MIF residues remain to be unknown. The possible induced conformational change of MIF during its binding with bioactive molecules, the role of key residues in the catalytic reactions and the interaction of MIF with different substrates, inhibitors need to be further investigated.Theoretical study at the atomic level was performed to tackle questions that beyond the reach of experiments. The importance of several key residues for the catalytic reactions as well as the specific recognition and binding affinity of MIF, MIF mutants with different inhibitors and substrates were further investigated. The conformational change of MIF during the complex formation was dynamically studied and the comparisons for the interactions of bioactive molecules with MIF and MIF mutants were also made.The interaction of eight hydroxycinnamate analogues, five substrate molecules, two ISO-1 analogues, five coumarin derivative molecules molecules with MIF and the interaction between eight hydroxycinnamate analogues and three MIF mutants, P1G; K32A, N97A were fully studied using molecular docking and molecular dynamics simulation.1. To elucidate the stereochemistry preference of MIF for (E)-2-fluoro-p-hydroxycinnamate and its analogues, the molecular dynamics simulations were performed on these six cinnamate analogues and the MM-PBSA analysis was also made for the evaluation of the binding free energy. The conformation of MIF is very steady and it shows no large change during the binding of cinnamate analogues. MIF has formed hydrogen bonds or salt bridge with cinnamate analogues mainly through residue Asn-97', Pro-1, Lys-32. The binding modes of E-ligands are much different from those of Z-ligands, i. e. E-ligands have hydrogen bonds with Pro-1 while no hydrogen bond was found between Z-ligands and Pro-1. This is the main cause of large difference in the binding affinities and the MM-PBSA analysis shows that the electrostatic interaction mostly contributes to this difference.2. To study the difference in the interaction of cinnamate analogues between MIF and its mutants and to investigate the mutational effects of MIF on its conformation, the complex structures of cinnamate analogues with P1G, K32A, NP7A mutants were constructed with molecular docking and these structures were then studied by molecular dynamics simulations. The hydrogen bond analysis revealed that the binding mode of E-ligands to MIF or P1G is different from that of Z-ligands. For E-ligand, it has a N-H…O hydrogen bond with Pro-1 of MIF or with Ile-64 of P1G; however, no obvious binding difference was found between Z-ligands and the proteins. There is no any hydrogen bond of Z-ligands formed with Pro-1 of MIF or with Ile-64 of P1G.. N97A mutant can form a hydrogen bond with both Eand Z-ligands via its residue Ile-64, while it have a salt bridge only with E-ligands through its residue Lys-32. Besides forming a O-H…O hydrogen bond with residue Asn-97' of K32A mutant, the E-ligands have a N-H…O hydrogen bond with Ile-64 of K32A mutant, however, the Z-ligands only have a O-H…O hydrogen bond with Asn-97' of K32A mutant. The calculated binding free energy difference shows that there still exist a difference in the inhibition between the E- and Z-ligands for P1G, K32A, N97A mutants. This lead us a suggestion that P1G, K32A, NP7A may still have certain stereochemistry preference for these analogues. Molecular dynamics simulations have been also performed on the trimer, monomer of MIF, N97A, K32A mutants and no large conformational change were found. The conformation shows some rigidity on the nanosecond time scale and it was not affected much by the residue mutations.3. To investigate the binding specificity of MIF with p-hydroxyphenylpyruvate(HPP), R-dopachrome(RDP), R- and S-dopachrome methyl ester(RDPM, SDPM), molecular docking was performed to construct their structures. The multiple molecular dynamics simulations were then carried out on these structures and MM-PBSA analysis was also performed to evaluate the binding free energy. HPP forms two hydrogen bonds with MIF, one O-H…O hydrogen bond with Asn-97' and one N-H…O hydrogen bond with Ile-64. There also exists a salt bridge between HPP and Lys-32 of MIF. RDP has two N-H…O hydrogen bonds with Pro-1 and Ile-64 of MIF. The N-H…N and O-H…O hydrogen bond are formed between SDPM and Asn-97', Pro-1 of MIF, However, only one N-H…O hydrogen bond with limited stability exists between RDPM and Pro-1 of MIF. MM-PBSA analysis gave a correct ranking binding affinity of these MIF substrates that RDPM＞HPP＞SDPM＞RDP. This order is in agreement with the experiment that, RDPM and SDPM are better substrates than RDP. The MM-PBSA analysis also revealed that the van der Waals interaction contributes the most part of the binding free energy and this interaction is of most significance to the whole interaction between MIF and these four substrates.4. Molecular dynamics simulations were performed on the complexes of MIF with two ISO-1 analogues(R1, S1) to investigate the detailed binding mode. MM-PBSA analysis was also carried out to evaluate the binding free energy of R-and S types of ISO-1. R1, S1 each has a O-H…O hydrogen bond with MIF. MM-PBSA analysis revealed that there is an obvious difference in the binding affinity between R and S ISO-1 and the electrostatic interactions are the predominant factor to this binding affinity difference.5. For a series of coumarin derivatives(A, B, C, D, E) as potent inhibitors of MIF, molecular docking was used to construct their complexes with MIF and molecular dynamics simulations were subsequently performed on these five complexes. Five coumarin derivative molecules all forms one O-H…O hydrogen bond with Asn-97' of MIF. Molecule A and E each has another N-H…O hydrogen bond with Ile-64, however, molecule B, C, D each forms another N-H…O hydrogen bond with Pro-1. These coumarin derivative molecules are surrounded by the hydrophobic residues and the hydrophobic force can enforce their binding affinities.The information obtained from this study could help to clarify enzymatic mechanism for MIF and may shed light on its enzymatic activity. It could provide instructive information for the structure-based drug design in the treatment of a variety of inflammatory and immune-related diseases associated with MIF.