Abstract:
Dynamic cell polarity is crucial for many cellular activities. In Myxococcus xanthus
MglA, a small Ras-like GTPase, and MglB, its GTPase activating protein (GAP), along
with RomR (response regulator domain) establish and regulate cell polarity. Recently
another small Ras-like GTPase SofG was discovered, which is critical for polar
localization of PilB and PilT, the proteins required for pili localization at the leading
pole. Both SofG and MglA work in synchrony to drive cell polarity in Myxococcus
xanthus. Towards understanding the molecular mechanism of SofG action, purification
of SofG was optimized, and biochemical characterization was carried out. SofG was
present as a homogenous monomer in solution and bound to GDP and GTP. Intrinsic
GTP hydrolysis of SofG was negligible. Based on sequence analysis, we hypothesized
that MglB could potentially act as a GAP for SofG too, and experimentally showed that
MglB increases the GTPase activity of SofG. Earlier work from the lab revealed that
MglB functions both as a GAP and a guanosine nucleotide exchange factor (GEF) for
MglA. However, our results showed that MglB did not function as a GEF for SofG and
did not interact with it in the GDP-bound conformation. The presence of a common
GAP for both SofG and MglA could potentially contribute to concerted regulatory
mechanisms of their GTPase activities, and mediate crosstalk between the two
GTPases within the cell.
Our sequence analysis of the MglB interacting interface also led to the discovery of a
novel catalytic motif in prokaryotic small Ras-like GTPases. Interestingly, the Walker
B aspartate, thought to be absent in prokaryotic small Ras-like GTPases, was located
within this newly identified motif. This was further validated experimentally by
mutational analysis and GTPase activity measurements.
