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Structural and biophysical insights into RomR, MglB, and MglC interactions involved in regulating cell polarity in Myxococcus xanthus
Cell polarity is an important phenomenon that helps in modulating many essential cellular functions, including motility in bacteria. In Myxococcus xanthus, cell polarity is mediated by a complex network of interacting proteins, including Ras-like GTPase protein Mgl (mutual gliding-motility protein) A, MglB, MglC, and RomRX (required for motility response regulator) complex. The interaction dynamics of these proteins are essential for the localization and switching of interacting partners. In this study, we performed a detailed interaction analysis to determine binding affinities and stoichiometry of the complexes involved in polarity reversal. We show that the RomR C-terminal helix, RomR371-420, alone is sufficient to bind MglC in 3:2 stoichiometry with a KD of 2.6 nM. A combination of AlphaFold3 structural modeling and site-directed mutagenesis experiments suggests that W394 in RomR is crucial for binding MglC. Using isothermal titration calorimetry experiments, we further show that MglB does not bind RomR371-420; however, interestingly, the MglB-MglC complex binds RomR371-420 with â�¼57-fold reduced affinity (KD â�¼150 nM), suggesting binding of MglB reduces the binding affinity of MglC toward RomR. Size-exclusion chromatography coupled with small-angle X-ray scattering (SEC-SAXS) analysis further supports that RomR371-420 exists as a homotrimer in solution and forms MglB-MglC-RomR371-420 complex with 4:2:3 stoichiometry. Using AlphaFold3 and SASREF to predict protein-protein complex structures, docking them into the SEC-SAXS generated envelope, we propose plausible models for MglC-RomR and MglB-MglC-RomR complexes. This study provides a basis to further study and decipher the role of these protein-protein interactions in polarity establishment and reversal.
HTML Summary of #3234 Structural and biophysical insights into RomR, MglB, and MglC interactions involved in regulating cell polarity in Myxococcus xanthus