Abstract:
Small Ras-like GTPases are known to act as molecular switches and regulate vital cellular processes across different kingdoms of species. In Myxococcus xanthus, two paralogous small Ras-like GTPases MglA and SofG act in concert to regulate its cellular polarity and motility. Earlier work from our lab has shown that MglB acts as an essential GTPase activating protein (GAP) and Guanine nucleotide exchange factor (GEF) for MglA. While SofG requires MglB for stimulating GTPase activity, it does not require MglB as a GEF. SofG has an additional C-terminal extension which was hypothesized to be responsible for its intrinsic GEF activity. Therefore, to investigate the role of C-terminal extension of SofG in GEF activity, C-terminal of SofG was introduced in the core of MglA and was called MglASGαCt. Biochemical characterization of MglASGαCt revealed that the GTP hydrolysis rate of MglASGαCt was enhanced in the presence of MglB compared to MglA and unlike MglA it was independent of MglB Ct-helix. Moreover, fluorescence-based assays revealed that MglASGαCt was capable of exchanging GDP to GTP suggesting that the C-terminal extension contributes to GEF activity. It was also determined that MglASGαCt was intrinsically bound to GDP. Crystallization screening to determine MglASGαCt structure was done to further validate these findings. Additionally, attempts to optimize purification of SofG was done by cloning it with SUMO-tag and C-terminal strep tag. Overall, this study provides insights into the mechanism of regulation of the GEF activity in SofG and the effects of having additional C-terminal extension on small- Ras like GTPases.
