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Summary: Prenylated Rab proteins exist in the cytosol as soluble, high-affinity complexes with GDI that need to be disrupted for membrane attachment and targeting of Rab proteins. The Legionella pneumophila protein DrrA displaces GDI from Rab1:GDI complexes, incorporating Rab1 into Legionella-containing vacuoles and activating Rab1 by exchanging GDP for GTP. Here, we present the crystal structure of a complex between the GEF domain of DrrA and Rab1 and a detailed kinetic analysis of this exchange. DrrA efficiently catalyzes nucleotide exchange and mimics the general nucleotide exchange mechanism of mammalian GEFs for Ras-like GTPases. We show that the GEF activity of DrrA is sufficient to displace prenylated Rab1 from the Rab1:GDI complex. Thus, apparent GDI displacement by DrrA is linked directly to nucleotide exchange, suggesting a basic model for GDI displacement and specificity of Rab localization that does not require discrete GDI displacement activity. [Copyright &y& Elsevier]

Members of the Ras superfamily of small GTPases are frequentlymutated in cancer. Therefore, inhibitors have been developed to addressthe acitivity of these GTPases by inhibiting their prenylating enzymesFTase, GGTase I, and RabGGTase. In contrast to FTase and GGTase I,only a handful of RabGGTase inhibitors have been developed. The mostactive RabGGTase inhibitor known until recently was an FTase inhibitorwhich hit RabGGTase as an off-target. We recently reported our effortsto tune the selectivity of these inhibitors toward RabGGTase. Herewe describe an extended set of selective inhibitors. The requirementsfor selective RabGGTase inhibitors are described in detail, guidedby multiple crystal structures. In order to relate in vitro and cellular activity, a high-throughput assay system to detect theattachment of [3H]geranylgeranyl groups to Rab was used.Selective RabGGTase inhibition allows the establishment of novel drugdiscovery programs aimed at the development of anticancer therapeutics. [ABSTRACT FROM AUTHOR]

Post-translational attachment of geranylgeranyl isoprenoids to Rab GTPases, the key organizers of intracellular vesicular transport, is essential for their function. Rab geranylgeranyl transferase (RabGGTase) is responsible for prenylation of Rab proteins. Recently, RabGGTase inhibitors have been proposed to be potential therapeutics for treatment of cancer and osteoporosis. However, the development of RabGGTase selective inhibitors is complicated by its structural and functional similarity to other protein prenyltransferases. Herein we report identification of the natural product psoromic acid (PA) that potently and selectively inhibits RabGGTase with an IC50 of 1.3 μM. Structure-activity relationship analysis suggested a minimal structure involving the depsidone core with a 3-hydroxyl and 4-aldehyde motif for binding to RabGGTase. Analysis of the crystal structure of the RabGGTase:PA complex revealed that PA forms largely hydrophobic interactions with the isoprenoid binding site of RabGGTase and that it attaches covalently to the N-terminus of the α subunit. We found that in contrast to other protein prenyltransferases, RabGGTase is autoinhibited through N-terminal αHis2 coordination with the catalytic zinc ion. Mutation of αHis dramatically enhances the reaction rate, indicating that the activity of RabGGTase is likely regulated in vivo. The covalent binding of PA to the N-terminus of the RabGGTase α subunit seems to potentiate its interaction with the active site and explains the selectivity of PA for RabGGTase. Therefore, psoromic acid provides a new starting point for the development of selective RabGGTase inhibitors. [ABSTRACT FROM AUTHOR]

In the course of Legionnaires' disease, the bacterium Legionella pneumophila affects the intracellular vesicular trafficking of infected eukaryotic cells by recruiting the small guanosine triphosphatase (GTPase) Rab1 to the cytosolic face of the Legionella-containing vacuole. In order to accomplish this, the Legionella protein DrrA contains a specific guanine nucleotide exchange activity for Rab1 activation that exchanges guanosine triphosphate (GTP) for guanosine diphosphate on Rab1. We found that the amino-terminal domain of DrrA possesses adenosine monophosphorytation (AMPylation) activity toward the switch II region of Rab1b, leading to posttranslational covalent modification of tyrosine 77. AMPylation of switch II by DrrA restricts the access of GTPase activating proteins, thereby rendering Rab1b constitutively active. [ABSTRACT FROM AUTHOR]

Summary Cell migration often involves the formation of sheet-like lamellipodia generated by branched actin filaments. The branches are initiated when Arp2/3 complex [ 1 ] is activated by WAVE regulatory complex (WRC) downstream of small GTPases of the Rac family [ 2 ]. Recent structural studies defined two independent Rac binding sites on WRC within the Sra-1/PIR121 subunit of the pentameric WRC [ 3, 4 ], but the functions of these sites in vivo have remained unknown. Here we dissect the mechanism of WRC activation and the in vivo relevance of distinct Rac binding sites on Sra-1, using CRISPR/Cas9-mediated gene disruption of Sra-1 and its paralog PIR121 in murine B16-F1 cells combined with Sra-1 mutant rescue. We show that the A site, positioned adjacent to the binding region of WAVE-WCA mediating actin and Arp2/3 complex binding, is the main site for allosteric activation of WRC. In contrast, the D site toward the C terminus is dispensable for WRC activation but required for optimal lamellipodium morphology and function. These results were confirmed in evolutionarily distant Dictyostelium cells. Moreover, the phenotype seen in D site mutants was recapitulated in Rac1 E31 and F37 mutants; we conclude these residues are important for Rac-D site interaction. Finally, constitutively activated WRC was able to induce lamellipodia even after both Rac interaction sites were lost, showing that Rac interaction is not essential for membrane recruitment. Our data establish that physical interaction with Rac is required for WRC activation, in particular through the A site, but is not mandatory for WRC accumulation in the lamellipodium. Graphical Abstract Highlights • WRC harbors two Rac binding sites, A and D, with distinct functions in vivo • A site is the major WRC activation site • Rac-D site interaction optimizes WAVE-mediated actin filament assembly • Both sites are obligatory for WRC activation, but not its lamellipodial accumulation Schaks et al. use gene disruption combined with re-expression of point mutants of the Sra-1/PIR121 subunit of mammalian WRC or the PIR ortholog in Dictyostelium to unravel differential functions of the two Rac interaction surfaces on WRC in vivo. These functions are crucial to both lamellipod/pseudopod protrusion and cell-migration efficiency. [ABSTRACT FROM AUTHOR]