Structure and Dynamics of Macrophage Infectivity Potentiator Proteins from Pathogenic Bacteria and Protozoans Bound to Fluorinated Pipecolic Acid Inhibitors

Macrophage infectivity potentiator (MIP) proteins, found in pro- and eukaryotic pathogens, influence microbial virulence, host cell infection, pathogen replication, and dissemination. MIPs share an FKBP (FK506 binding protein)-like prolyl-<i>cis/trans</i>-isomerase domain, making them attractive targets for inhibitor development. We determined high-resolution crystal structures of <i>Burkholderia pseudomallei</i> and <i>Trypanosoma cruzi</i> MIPs in complex with fluorinated pipecolic acid inhibitors. The inhibitor binding profiles in solution were compared across <i>B. pseudomallei</i>, <i>T. cruzi</i>, and <i>Legionella pneumophila</i> MIPs using ¹H, ¹⁵N, and ¹⁹F NMR spectroscopy. Demonstrating the versatility of fluorinated ligands for characterizing inhibitor complexes, ¹⁹F NMR spectroscopy identified differences in ligand binding dynamics across MIPs. EPR spectroscopy and SAXS further revealed inhibitor-induced global structural changes in homodimeric <i>L. pneumophila</i> MIP. This study demonstrates the importance of integrating diverse methods to probe protein dynamics and provides a foundation for optimizing MIP-targeted inhibitors in this structurally conserved yet dynamically variable protein family.