Nanosecond Dynamics Regulate the MIF‐Induced Activity of CD74

Abstract

Macrophage migration inhibitory factor (MIF) activates CD74, whichl eads to severe disorders including inflammation, autoimmune diseases and cancer under pathological conditions.M olecular dynamics (MD) simulations up to one microsecond revealed dynamical correlation between aresidue located at the opening of one end of the MIF solvent channel, previously thought to be ac onsequence of homotrimerization, and residues in ad istal region responsible for CD74 activation. Experiments verified the allosteric regulatory site and identified apathway to this site via the MIF b-strands. The reported findings providef undamental insights on ad ynamic mechanism that controls the MIF-induced activation of CD74.MIF is ap ro-inflammatory cytokine with regulatory functions that control cell fate.I np athological conditions, such as cancer, MIF serves to manipulate the immune system to benefit malignant cells. [1] Thec ascade of signaling events that promote tumor growth and metastasis is initiated by the MIF-induced activation of the cell surface receptor CD74. [2] MIF is also an on-cognate,p artial agonist of CXCR2 and CXCR4. [3] Despite the extensive understanding of the MIF role in immune system physiology and pathology,westill lack af undamental understanding of all aspects of protein structure and activity.G eneration of ac o-crystal or NMR structure between MIF and its primary receptor CD74 has been difficult to obtain due to the increased flexibility of CD74. [4] However,i nsights into the MIF/CD74 interaction can be obtained using other methods that are focused on understanding the intermolecular mechanism of CD74 activation by MIF.N umerous crystal structures of wild-type and mutants of MIF have been determined, as well as MIF cocrystal structures with substrates,c ompetitive,n on-competitive,and covalent inhibitors. [5][6][7][8][9][10] No significant changes in the MIF tertiary or quaternary structures have been observed, other than movements of side chains.S uch findings give the impression that local flexibility [11] is important for regulation of MIF biological functions,i ncluding activation of the cell surface receptor CD74. TheMIF surface residues responsible for activation of CD74 were mapped by epitope scanning [12] and alanine-scanning mutagenesis. [9] To probe whether local flexibility of the MIF trimer has any role in CD74 activation, we employed atomistic molecular dynamics simulations of wild-type human MIF over 1 ms. Our findings show that the averaged root-mean-square fluctuation (RMSF) over all residues is 0.90 indicating that the system remains in at ightly clustered ensemble (Supporting Information, Figure S1a,b). Correlations between fluctuations in the displacement of Ca of pairs of residues i and j were identified with the generalized correlation matrix, with entries C ij . This approach provides an improvement over the calculation of covariance matrices and linear correlations. [13] Theapproximate threefold symmetry of the protein is reflected in the correlation plot where each boxed region repre...

Designed CXCR4 mimic acts as a soluble chemokine receptor that blocks atherogenic inflammation by agonist-specific targeting

“…A molecular explanation for this selectivity comes from experiments mapping the MIF binding site by MIF peptide fragments and a site-specific mutant. msR4M-L1 targets MIF region 54-80, a part of the N-like loop known to mediate MIF/CXCR4 binding, but not involved in MIF/CD74 binding, in line with data showing that the tautomerase site of MIF, Tyr-100, and residues 80-87 determine the MIF/CD74 binding interface 21,22,59 . Importantly, binding selectivity of msR4M-L1 for MIF versus CXCL12 was functionally paralleled in a number of inflammation-and atherosclerosisrelevant cell systems, i.e.…”

“…8). MIF residues 80-87 and Tyr-100 are specific determinants of the interaction between MIF and CD74, whereas Pro-2 of MIF contributes to both MIF/CD74 binding and the MIF/CXCR4 interface 21,22,25 . We therefore also applied FP and MST in a binding-competition approach to experimentally confirm that msR4M-L1 does not interfere with MIF binding to its receptor CD74, which mediates MIF's cardioprotective activity 15 .…”

“…On the other hand, peptide segment 6-23, which is outside the core region, or 62-80 which is situated at the far C-terminal end of the core, did not bind to MIF (app. K D Fluos-msR4M-L1/MIF [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] Fig. 10; Supplementary Fig.…”

“…We recently elucidated the structural determinants of the binding interface between MIF and its receptors. Binding of MIF to CD74 involves MIF residues Pro-2, 80-87, and Tyr-100, while MIF binding to CXCR2 requires a pseudo-ELR motif, similar to CXCL8 19,[21][22][23] . In contrast, the cognate CXCR4 ligand CXCL12/ stromal cell-derived factor-1alpha (SDF-1α) is an ELR-negative chemokine and the CXCL12/CXCR4 interface involves the receptor N-terminus and the RFFESH motif of CXCL12 at site 1 and the chemokine N-terminus and an intramembrane receptor groove at site 2.…”

Structural Plasticity in the C-Terminal Region of Macrophage Migration Inhibitory Factor-2 Is Associated with an Induced Fit Mechanism for a Selective Inhibitor

“…9,11–13 Recent mechanistic insights into the MIF-1-induced activation of CD74 showed that the activation process is dynamically controlled by an allosteric site that is found on one side of the solvent channel. 14 Whether a similar allosteric site exists for MIF-2 has yet to be explored. MIF-1 also activates chemokine receptors CXCR2 and CXCR4 leading to cell cycle arrest, integrin activation, chemotaxis, and calcium influx.…”