Mechanism of GTPase activation of a prokaryotic small Ras-like GTPase MglA by an asymmetrically interacting MglB dimer.

Cell polarity oscillations in Myxococcus xanthus motility are driven by a prokaryotic small Ras-like GTPase, mutual gliding protein A (MglA), which switches from one cell pole to the other in response to extracellular signals. MglA dynamics is regulated by MglB, which functions both as a GTPase activating protein (GAP) and a guanine nucleotide exchange factor (GEF) for MglA. With an aim to dissect the asymmetric role of the two MglB protomers in the dual GAP and GEF activities, we generated a functional MglAB complex by coexpressing MglB with a linked construct of MglA and MglB. This strategy enabled us to generate mutations of individual MglB protomers (MglB or MglB linked to MglA) and delineate their role in GEF and GAP activities. We establish that the C-terminal helix of MglB, but not MglB, stimulates nucleotide exchange through a site away from the nucleotide-binding pocket, confirming an allosteric mechanism. Interaction between the N-terminal β-strand of MglB and β of MglA is essential for the optimal GEF activity of MglB. Specific residues of MglB which interact with Switch-I of MglA, partially contribute to its GAP activity. Thus, the role of the MglB protomer in the GAP activity of MglB is limited to restricting the conformation of MglA active site loops. The direct demonstration of the allosteric mechanism of GEF action provides us new insights into the regulation of small Ras-like GTPases, a feature potentially present in many uncharacterized GEFs.