A structurally preserved allosteric site in the MIF superfamily affects enzymatic activity and CD74 activation in D-dopachrome tautomerase

The macrophage migration inhibitory factor (MIF) family of cytokines contains multiple ligand-binding sites and mediates immunomodulatory processes through an undefined mechanism(s). Previously, we reported a dynamic relay connecting the MIF catalytic site to an allosteric site at its solvent channel. Despite structural and functional similarity, the MIF homolog D-dopachrome tautomerase (also called MIF-2) has low sequence identity (35%), prompting the question of whether this dynamic regulatory network is conserved. Here, we establish the structural basis of an allosteric site in MIF-2, showing with solution NMR that dynamic communication is preserved in MIF-2 despite differences in the primary sequence. X-ray crystallography and NMR detail the structural consequences of perturbing residues in this pathway, which include conformational changes surrounding the allosteric site, despite global preservation of the MIF-2 fold. Molecular simulations reveal MIF-2 to contain a comparable hydrogen bond network to that of MIF, which was previously hypothesized to influence catalytic activity by modulating the strength of allosteric coupling. Disruption of the allosteric relay by mutagenesis also attenuates MIF-2 enzymatic activity in vitro and the activation of the cluster of differentiation 74 receptor in vivo , highlighting a conserved point of control for nonoverlapping functions in the MIF superfamily.

Automated summary

Despite the low sequence identity, the study revealed a conserved dynamic regulatory network between MIF and MIF-2, with the structural basis of an allosteric site in MIF-2 being established. Molecular simulations showed a comparable hydrogen bond network in MIF-2, suggesting its influence on catalytic activity through modulating allosteric coupling. Disruption of the allosteric relay in MIF-2 attenuated enzymatic activity in vitro and receptor activation in vivo, indicating a conserved point of control for nonoverlapping functions in the MIF superfamily.