The well-studied members of the Trio family of proteins are Trio and kalirin in vertebrates, UNC-73 in Caenorhabditis elegans and Trio in Drosophila. Trio proteins are key regulators of cell morphogenesis and migration, tissue organization, and secretion and protein trafficking in many biological contexts. Recent discoveries have linked Trio and kalirin to human disease, including neurological disorders and cancer. The genes for Trio family proteins encode a series of large multidomain proteins with up to three catalytic activities and multiple scaffolding and protein–protein interaction domains. As such, Trio family proteins engage a wide array of cell surface receptors, substrates and interaction partners to coordinate changes in cytoskeletal regulatory and protein trafficking pathways. We provide a comprehensive review of the specific mechanisms by which Trio family proteins carry out their functions in cells, highlight the biological and cellular contexts in which they occur, and relate how alterations in these functions contribute to human disease.

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Rho family GTPases regulate an array of cellular processes and are often modulated by pathogens to promote infection. Here, we identify a cryptic guanine nucleotide exchange factor (GEF) domain in the OtDUB protein encoded by the pathogenic bacteriumOrientia tsutsugamushi. A proteomics-based OtDUB interaction screen identified numerous potential host interactors, including the Rho GTPases Rac1 and Cdc42. We discovered a domain in OtDUB with Rac1/Cdc42 GEF activity (OtDUBGEF), with higher activity toward Rac1 in vitro. While this GEF bears no obvious sequence similarity to known GEFs, crystal structures of OtDUBGEFalone (3.0 Å) and complexed with Rac1 (1.7 Å) reveal striking convergent evolution, with a unique topology, on a V-shaped bacterial GEF fold shared with other bacterial GEF domains. Structure-guided mutational analyses identified residues critical for activity and a mechanism for nucleotide displacement. Ectopic expression of OtDUB activates Rac1 preferentially in cells, and expression of the OtDUBGEFalone alters cell morphology. Cumulatively, this work reveals a bacterial GEF within the multifunctional OtDUB that co-opts host Rac1 signaling to induce changes in cytoskeletal structure.

17Rho family GTPases regulate an array of cellular processes and are often modulated by 18 pathogens to promote infection. Here, we identify a cryptic guanine nucleotide exchange factor 19 (GEF) domain in the OtDUB protein encoded by the pathogenic bacterium Orientia 20 tsutsugamushi. A proteomics-based OtDUB interaction screen identified numerous potential host 21 interactors, including the Rho-GTPases Rac1 and Cdc42. We discovered a new domain in 22OtDUB with Rac1/Cdc42 GEF activity (OtDUB GEF ), with higher activity toward Rac1 in vitro. 23While this GEF bears no obvious sequence similarity to known GEFs, crystal structures of 24 OtDUB GEF alone (3.0 Å) and complexed with Rac1 (1.7 Å) reveal striking convergent evolution, 25 with a distinct topology, on a V-shaped bacterial GEF fold shared with other bacterial GEF 26 domains. Structure-guided mutational analyses identified residues critical for activity and a novel 27 mechanism for nucleotide displacement. Ectopic expression of OtDUB activates Rac1 28 preferentially in cells, and expression of the OtDUB GEF alone alters cell morphology. 29Cumulatively, this work reveals a novel bacterial GEF within the multifunctional OtDUB that 30 co-opts host Rac1 signaling to evoke changes in cytoskeletal structure. 31 motif important for folding and structural integrity) or the SopE family (SopE, SopE2, and 55 BopE). These bacterial effectors share no sequence or structural homology to eukaryotic Rho 56 GEFs, which predominantly belong to the Dbl homology (DH) family of GEFs typically formed 57 by a six-helix bundle with an elongated, kinked "chaise lounge" fold. 8,9 Rather, bacterial effector 58GEFs adopt a characteristic compact V-shaped fold, yet activate the Rho GTPases via the same 59 contact regions in the GTPases that are crucial for nucleotide exchange by DH-family GEFs. 10 60While substantial effort has been exerted in detailing the molecular determinants of bacterial 61 GEF activities and specificities, no bacterial effector GEFs have been identified outside of the 62 WxxxE/SopE-like families. 63Recently, we identified and characterized a putative effector protein, OtDUB, from the 64 obligate intracellular bacterium that causes scrub typhus, Orientia tsutsugamushi. Despite 65 extensive characterization of the OtDUB deubiquitylase (DUB) domain (residues 1-259), the 66 function of the extensive C-terminal region, encompassing more than 1000 amino acids, 67 remained elusive. 11 Here, we report that the OtDUB encodes a novel GEF domain. Using 68 biochemical, structural, and cellular methods, we demonstrate that the OtDUB GEF predominantly 69 activates Rac1 in vitro and in vivo, and that its three-dimensional structure differs drastically in 70 topological and helical arrangement from both the WxxxE and SopE effector GEFs. We further 71 determined the OtDUB GEF :Rac1 complex crystal structure, which reveals that despite its 72 divergence in primary sequence and topology, the OtDUB GEF has convergently evolved the V-73 shaped fold of bacterial GEFs and interacts with ...

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