In Myxococcus xanthus, directed movement is controlled by pole-to-pole oscillations of the small GTPase MglA and its GAP MglB. Direction reversals require that MglA is inactivated by MglB, yet paradoxically MglA and MglB are located at opposite poles at reversal initiation. Here we report the complete MglA/MglB structural cycle combined to GAP kinetics and in vivo motility assays, which uncovers that MglA is a three-state GTPase and suggests a molecular mechanism for concerted MglA/MglB relocalizations. We show that MglA has an atypical GTP-bound state (MglA-GTP*) that is refractory to MglB and is re-sensitized by a feedback mechanism operated by MglA-GDP. By identifying and mutating the pole-binding region of MglB, we then provide evidence that the MglA-GTP* state exists in vivo. These data support a model in which MglA-GDP acts as a soluble messenger to convert polar MglA-GTP* into a diffusible MglA-GTP species that re-localizes to the opposite pole during reversals.

Mutations in the Parkinson's disease (PD)-associated protein leucine-rich repeat kinase 2 (LRRK2) commonly lead to a reduction of GTPase activity and increase in kinase activity. Therefore, strategies for drug development have mainly been focusing on the design of LRRK2 kinase inhibitors. We recently showed that the central RocCOR domains (Roc: Ras of complex proteins; COR: C-terminal of Roc) of a bacterial LRRK2 homolog cycle between a dimeric and monomeric form concomitant with GTP binding and hydrolysis. PD-associated mutations can slow down GTP hydrolysis by stabilizing the protein in its dimeric form. Here, we report the identification of two Nanobodies (NbRoco1 and NbRoco2) that bind the bacterial Roco protein (CtRoco) in a conformation-specific way, with a preference for the GTP-bound state. NbRoco1 considerably increases the GTP turnover rate of CtRoco and reverts the decrease in GTPase activity caused by a PD-analogous mutation. We show that NbRoco1 exerts its effect by allosterically interfering with the CtRoco dimer–monomer cycle through the destabilization of the dimeric form. Hence, we provide the first proof of principle that allosteric modulation of the RocCOR dimer–monomer cycle can alter its GTPase activity, which might present a potential novel strategy to overcome the effect of LRRK2 PD mutations.

22In Myxococcus xanthus, directed movement is controlled by inter-dependent pole-to-pole 23 oscillations of the small GTPase MglA, its GAP MglB and the RomR protein. However, these 24 proteins have strikingly different oscillatory regimes such that MglA is segregated from MglB 25 and RomR at reversal activation. The molecular mechanism whereby information is 26 exchanged between the lagging and leading poles resulting in MglA detachment from the 27 leading pole during reversals has remained unknown. Here, we show that MglA has two GTP-28 bound forms, one of which is insensitive to MglB (MglA-GTP*) and is re-sensitized to MglB 29 by a feedback mechanism operated by MglA-GDP. By identifying the region of MglB that is 30 critical for its association to the lagging pole, we demonstrate that MglA-GTP* is functional 31 in vivo. These data suggest that MglA-GDP acts as a soluble messenger to convert polar 32MglA-GTP* into a diffusible MglA-GTP species, explaining MglA re-localization to the 33 opposite pole during reversals. 34 35 36 42GTPase-activating proteins or GAPs, which stimulate GTP hydrolysis (reviewed in (Cherfils 43 and Zeghouf, 2013)). Polarity of migration controlled by small GTPases in prominent in 44 eukaryotes (reviewed in (Charest and Firtel, 2007)), and has also been discovered in 45