Understanding the mechanism of regulation of a small Ras-like GTPase, MglA, driving Myxococcus xanthus motility

Abstract

Polarity reversal in the soil bacterium Myxococcus xanthus is regulated by a cascade of proteins depending on extracellular signals. MglA, a prokaryotic small Ras-like GTPase, is the master regulator of this motility. MglA GTP hydrolysis is accelerated by its cognate GTPase activating protein (GAP), MglB which also acts as the Guanine nucleotide exchange factor (GEF). My work aims to dissect this dual GAP and GEF activity of MglB in light of the asymmetric interaction of MglB dimer with MglA. We observe that the C-terminal helix of the MglB1 protomer interacts with MglA opposite the nucleotide-binding pocket, allosterically stimulating nucleotide exchange. The GAP activity is driven by the MglB dimeric interface, which orients the MglA catalytic residues. Interaction of MglB orients a non-canonical Walker B motif in MglA important for nucleotide binding. We also characterize RomY, a newly identified co-GAP of MglB. RomR-RomX, a complex of two proteins, has also been shown to act as GEF for MglA, making it interesting to understand the regulation of these two different GEF modules. Domain-wise characterisation of RomR shows the N-terminal receiver domain interacting with RomX and consequently, we identify the interface. The crystal structure of RomX reveals it to be an alpha- helical protein, strikingly similar to the stalk domain of the GTPase atlastin. We observe the GEF activity of RomX without any contribution from the RomR receiver domain. We compare this with the MglB GEF activity, which aids in distinguishing the mechanisms of MglA activation by MglB and RomR-RomX. RomR has also been shown to interact with MglC, which also interacts with MglB. We observe that the C-terminal domain of RomR interacts with MglC, which plays a role in recruiting MglB. Fitting all these pieces into the puzzle, we finally propose an updated model of the mechanism of cell polarity maintenance and reversal, characterizing Myxococcus xanthus motility.