The Capping Domain in RalF Regulates Effector Functions

Abstract

The Legionella pneumophila effector protein RalF functions as a guanine nucleotide exchange factor (GEF) that activates the host small GTPase protein ADP-ribosylation factor (Arf), and recruits this host protein to the vacuoles in which this pathogen resides. GEF activity is conferred by the Sec7 domain located in the N-terminal region of RalF. Structural studies indicate that the C-terminal region of RalF makes contacts with residues in the Sec7 domain important for Arf interactions. Theoretically, the C-terminal region of RalF could prevent nucleotide exchange activity by blocking the ability of Arf to interact with the Sec7 domain. For this reason, the C-terminal region of RalF has been termed a capping domain. Here, the role of the RalF capping domain was investigated by comparing biochemical and effector activities mediated by this domain in both the Legionella RalF protein (LpRalF) and in a RalF ortholog isolated from the unrelated intracellular pathogen Rickettsia prowazekii (RpRalF). These data indicate that both RalF proteins contain a functional Sec7 domain and that the capping domain regulates RalF GEF activity. The capping domain has intrinsic determinants that mediate localization of the RalF protein inside of host cells and confer distinct effector activities. Localization mediated by the capping domain of LpRalF enables the GEF to modulate membrane transport in the secretory pathway, whereas, the capping domain of RpRalF enables this bacterial GEF to modulate actin dynamics occurring near the plasma membrane. Thus, these data reveal that divergence in the function of the C-terminal capping domain alters the in vivo functions of the RalF proteins.

Toxicity and SidJ-Mediated Suppression of Toxicity Require Distinct Regions in the SidE Family of Legionella pneumophila Effectors

“…Several L. pneumophila effectors have been found to have enzymatic activities that are regulated through autoinhibition. The GEF activity of RalF is autoinhibited by a C-terminal domain that functions as a cap that senses host membranes (48)(49)(50), and VipD is a phospholipase that is autoinhibited by an amino-terminal domain that binds to the host protein Rab5 (51)(52)(53). Thus, future studies on the mechanism by which SidJ regulates SidE function could reveal additional strategies by which the activities of L. pneumophila effectors are controlled spatially and temporally during infection.…”

Allosteric regulation of Arf GTPases and their GEFs at the membrane interface

“…67 Both bacterial ArfGEFs are strongly activated by membranes, and the membrane-binding site is identical to the elements in the autoinhibitory domain that blocks the Arf-binding site. 68,69 Thus, activation of Arf GTPases by RalF strictly depends on its recruitment to membranes. Legionella RalF produces Arf-GTP at the surface of the Legionella-containing vacuole where the pathogen hides and replicates; 65 however Rickettsia replicates in the host cytosol and therefore should use RalF for different functions.…”

Creating a customized intracellular niche: subversion of host cell signaling byLegionellatype IV secretion system effectors

“…3B). Control of RalF activity is exerted by an internal so-called capping domain, which localizes the effector to the LCV and only upon membrane contact opens up access to its Sec7-homology domain (Alix et al 2012). At the LCV, ARF1 facilitates efficient fusion of ER-derived vesicles (Robinson and Roy 2006).…”