Science 358, 528–531 (2017)

Credit: AAAS

Vibrio cholerae and other pathogenic bacteria encode multifunctional autoprocessing repeats-in-toxin (MARTX) proteins that insert into the host cell membrane and release their multiple effector domains into the cytoplasm. One of these MARTX effectors, the Rho GTPase inactivation domain (RID), decreases the amount of active GTP-bound Rho GTPases in host cells through a previously unknown mechanism. Zhou et al. determined that V. cholerae RID bound to Rac1, a Rho GTPase family member, trapping it at the cell membrane; a gel band shift indicated a post-translational modification of Rac1. The RID crystal structure revealed a bipartite structure with a membrane-binding N lobe and a catalytic C lobe similar to the human fatty acyltransferase HRASLS3. In line with fatty acyltransferase activity, RID also bound palmitoyl coenzyme A. Click chemistry and in vitro acylation assays provided further support that RID is a long-chain fatty acyltransferase and that Rac1 prenylation is a prerequisite for this modification. Mutagenesis and MS analysis revealed that RID catalyzes the Nɛ-fatty acylation of lysines in the C-terminal polybasic region of Rac1. Fatty acylation of Rac1 inhibited its activation by guanine nucleotide exchange factors and blocked downstream signaling. These structural and functional insights into the RID Nɛ-fatty acylation mechanism may be useful for future drug design.