A bipartite, low-affinity roadblock domain-containing GAP complex regulates bacterial front-rear polarity

Abstract

The Ras-like GTPase MglA is a key regulator of front-rear polarity in the rod-shaped Myxococcus xanthus cells. MglA-GTP localizes to the leading cell pole and stimulates assembly of the two machineries for type IV pili-dependent motility and gliding motility. MglA-GTP localization is spatially constrained by its cognate GEF, the RomR/RomX complex, and GAP, the MglB Roadblock-domain protein. Paradoxically, RomR/RomX and MglB localize similarly with low and high concentrations at the leading and lagging poles, respectively. Yet, GEF activity dominates at the leading and GAP activity at the lagging pole by unknown mechanisms. Here, we identify RomY and show that it stimulates MglB GAP activity. The MglB/RomY interaction is low affinity, restricting formation of the bipartite MglB/RomY GAP complex almost exclusively to the lagging pole with the high MglB concentration. Our data support a model wherein RomY, by forming a low-affinity complex with MglB, ensures that the high MglB/RomY GAP activity is confined to the lagging pole where it dominates and outcompetes the GEF activity of the RomR/RomX complex. Thereby, MglA-GTP localization is constrained to the leading pole establishing front-rear polarity.

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

“…Similarly, it is not known how Rom-C brings about the polar localization of RomR, and how RomR stimulates its polar binding. Second, experimental evidence and AlphaFold-Multimer structural models support that MglB binds its co-GAP RomY with low affinity on the two-helix side 28 . We, therefore, suggest that the RomR/MglC/MglB complex at the lagging pole also contains RomY forming a RomR/MglC/MglB/RomY complex.…”

“…Together these interactions have been suggested to result in the system's emergent properties (Fig. 1AB) 17,28 . Briefly, at the pole with the highest RomR concentration, the RomR/MglB positive feedback establishes a pole with high concentrations of RomR/RomX and MglB/RomY.…”

“…1A). The homodimeric roadblock domain protein MglB alone has GAP activity and together with its low-affinity co-GAP RomY, forms the MglB/RomY complex, which is the active GAP in vivo [26][27][28] . RomX alone has GEF activity and forms the RomR/RomX complex, the active GEF in vivo that also serves as a polar recruitment factor for MglA-GTP 29 .…”

“…Experiments and mathematical modelling have uncovered an intricate set of regulatory interactions between the proteins of the polarity module 17,[26][27][28][29][30][31][32] (Fig. 1B).…”

“…Additionally, RomR directly recruits RomX to form the RomR/RomX GEF complex. High concentrations of polar MglB stimulate polar recruitment of its low-affinity interaction partner RomY 28 . At the RomR node of the RomR/MglB positive feedback, RomR/RomX promotes MglA-GTP polar recruitment (Fig.…”