Structural basis of Mycobacterium Fluoroquinolone Resistance Protein D (MfpD), a versatile pathogeny protein from the mfp conservon of Mycobacterium tuberculosis.

The mfp conservon of Mycobacterium tuberculosis has been associated with fluoroquinolone resistance and encodes five conserved proteins, including the small GTPase MfpB and its regulatory partner MfpD. In this study, we combined phylogenetic, structural, and biophysical approaches to define the molecular basis of MfpD function. MfpD adopts a Roadblock/LC7-like α/β fold and forms a stable dimer in solution, with hydrophobic α2-helix interactions stabilizing the interface. Additional biophysical analyses and AlphaFold3 modeling suggest that MfpD may promote GTP hydrolysis by MfpB through a noncanonical Switch I-dependent mechanism. These findings establish the first structural framework for MfpD-MfpB interactions, building on previously identified in vitro catalytic properties and proposing new insights into MfpD's non-catalytic pathogenesis activity of MfpD in macrophages.