The Brucella TIR domain containing proteins BtpA and BtpB have a structural WxxxE motif important for protection...

The TIR domain-containing proteins BtpA/Btp1/TcpB and BtpB are translocated into host cells by the facultative intracellular bacterial pathogen Brucella. Here, they interfere with Toll like receptor signalling to temper the host inflammatory response. BtpA has also been found to modulate microtubule dynamics. In both proteins we identified a WxxxE motif, previously shown to be an essential structural component in a family of bacterial type III secretion system effectors that modulate host actin dynamics by functioning as guanine nucleotide exchange factors of host GTPases. We analysed a role for the WxxxE motif in association of BtpA and BtpB with the cytoskeleton.

Unlike BtpA, ectopically expressed BtpB did not show a tubular localisation, but was found ubiquitously in the cytoplasm and the nucleus, and often appeared in discrete punctae in HeLa cells. BtpB was able to protect microtubules from drug-induced destabilisation similar to BtpA. The WxxxE motif was important for the ability of BtpA and BtpB to protect microtubules against destabilising drugs. Surprisingly, ectopic expression of BtpA, although not BtpB, in HeLa cells induced the formation of filopodia. This process was invariably dependent of the WxxxE motif. Our recent resolution of the crystal structure of the BtpA TIR domain reveals that the motif positions a glycine residue that has previously been shown to be essential for interaction of BtpA with microtubules.

Our results suggest a structural role for the WxxxE motif in the association of BtpA and BtpB with microtubules, as with the WxxxE GEF family proteins where the motif positions an adjacent catalytic loop important for interaction with specific Rho GTPases. In addition, the ability of ectopically expressed BtpA to induce filopodia in a WxxxE-dependent manner suggests a novel property for BtpA. A conserved WxxxE motif is found in most bacterial and several eukaryotic TIR domain proteins. Despite the similarity between ectopically expressed BtpA and WxxxE GEFs to modulate host actin dynamics, our results suggest that BtpA is not part of this WxxxE GEF family. The WxxxE motif may therefore be a more common structural motif than thus far described. BtpA may provide clues to cross-talk between the TLR and GTPase signalling pathways.