Biochemical characterization of two prokaryotic small Ras-like GTPases and their common effector

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.