Structural characterization of Myxococcus xanthus MglC, a component of the polarity control system, and its interactions with its paralog MglB

Abstract

The δ-proteobacteria Myxococcus xanthus displays social (S) and adventurous (A) motilities, which require pole-to-pole reversal of the motility regulator proteins. M utual gl iding motility protein C (MglC), a paralog of GTPase-activating protein M utual gl iding motility protein B (MglB), is a member of the polarity module involved in regulating motility. However, little is known about the structure and function of MglC. Here, we determined ∼1.85 Å resolution crystal structure of MglC using Se lenomethionine S ingle-wavelength a nomalous d iffraction. The crystal structure revealed that, despite sharing <9% sequence identity, both MglB and MglC adopt a R egulatory L ight C hain 7 family fold. However, MglC has a distinct ∼30° to 40° shift in the orientation of the functionally important α2 helix compared with other structural homologs. Using isothermal titration calorimetry and size-exclusion chromatography, we show that MglC binds MglB in 2:4 stoichiometry with submicromolar range dissociation constant. Using small-angle X-ray scattering and molecular docking studies, we show that the MglBC complex consists of a MglC homodimer sandwiched between two homodimers of MglB. A combination of size-exclusion chromatography and site-directed mutagenesis studies confirmed the MglBC interacting interface obtained by molecular docking studies. Finally, we show that the C-terminal region of MglB, crucial for binding its established partner MglA, is not required for binding MglC. These studies suggest that the MglB uses distinct interfaces to bind MglA and MglC. Based on these data, we propose a model suggesting a new role for MglC in polarity reversal in M. xanthus .

Molecular basis and design principles of a system for switchable front-rear polarity and directional migration

“…A structural model of the RomR/MglC/MglB complex To gain structural insights into the RomR/MglC/MglB complex, we used structural information, our functional data and AlphaFold-Multimer structural predictions to model this complex. The AlphaFold-Multimer models of the MglB dimer and MglC dimer were predicted with high confidence and agreed well with the crystallographic structures 35,40,41 (Fig. S7ABC), documenting the validity of the structural predictions.…”

“…The MglC homodimer's structure is similar to that of MglB with two-helix and four-helix sides (Fig. 4A) 35 . Based on conservation, McLoon et al reported the MglC F25A D26A I28A variant (henceforth MglC FDI ) with substitutions on the two-helix side (Fig.…”

“…To elucidate the structural basis for the RomR→MglC→MglB interactions, we took advantage of structural information for MglA, MglB and MglC 35,[40][41][42] . Each MglB protomer in the homodimer consists of a five-stranded β-sheet sandwiched between the α2-helix and the α1/α3-helices.…”

“…Among the interactions of the proteins of the polarity module, the positive feedback of RomR on itself, the RomR/MglB positive feedback, and the inhibitory effect of MglA-GTP on this positive feedback are poorly understood. MglC is also a homodimeric roadblock domain protein [35][36][37] and is involved in cell polarity regulation by an unknown mechanism 36 . Because MglC interacts with RomR and MglB 35,36 , MglC was a candidate for acting in the RomR/MglB positive feedback.…”

“…MglC is also a homodimeric roadblock domain protein [35][36][37] and is involved in cell polarity regulation by an unknown mechanism 36 . Because MglC interacts with RomR and MglB 35,36 , MglC was a candidate for acting in the RomR/MglB positive feedback. Here, we show that MglC forms a complex with RomR and MglB, thereby establishing a RomR/MglC/MglB positive feedback and that MglA-GTP inhibits this positive feedback by breaking the interaction between the MglC and MglB roadblock domain proteins.…”