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. 2007 May 18;368(5):1307-20.
doi: 10.1016/j.jmb.2007.02.060. Epub 2007 Feb 22.

The DH and PH domains of Trio coordinately engage Rho GTPases for their efficient activation

Mariya K Chhatriwala  1 , Laurie Betts, David K Worthylake, John Sondek
Affiliations

The DH and PH domains of Trio coordinately engage Rho GTPases for their efficient activation

Mariya K Chhatriwala et al. J Mol Biol. .

Abstract

Rho-family GTPases are activated by the exchange of bound GDP for GTP, a process that is catalyzed by Dbl-family guanine nucleotide exchange factors (GEFs). The catalytic unit of Dbl-family GEFs consists of a Dbl homology (DH) domain followed almost invariantly by a pleckstrin-homology (PH) domain. The majority of the catalytic interface forms between the switch regions of the GTPase and the DH domain, but full catalytic activity often requires the associated PH domain. Although PH domains are usually characterized as lipid-binding regions, they also participate in protein-protein interactions. For example, the DH-associated PH domain of Dbs must contact its cognate GTPases for efficient exchange. Similarly, the N-terminal DH/PH fragment of Trio, which catalyzes exchange on both Rac1 and RhoG, is fourfold more active in vitro than the isolated DH domain. Given continued uncertainty regarding functional roles of DH-associated PH domains, we have undertaken structural and functional analyses of the N-terminal DH/PH cassette of Trio. The crystal structure of this fragment of Trio bound to nucleotide-depleted Rac1 highlights the engagement of the PH domain with Rac1 and substitution of residues involved in this interface substantially diminishes activation of Rac1 and RhoG. Also, these mutations significantly reduce the ability of full-length Trio to induce neurite outgrowth dependent on RhoG activation in PC-12 cells. Overall, these studies substantiate a general role for DH-associated PH domains in engaging Rho GTPases directly for efficient guanine nucleotide exchange and support a parsimonious explanation for the essentially invariant linkage between DH and PH domains.

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Figures

Figure 1
Figure 1
Trio-related GEFs. (a) Crystallized fragment of Trio is highlighted within the domain architecture of full-length Trio. (b) Residues of Dbs (arrows) that mediate contacts between its N-terminal PH domain and cognate GTPases are conserved in other Dbl-family GEFs, including Trio. The relative position of these residues in context of the DH/PH cassette is indicated in Figure 3.
Figure 2
Figure 2
Crystal structure of the DH/PH fragment of Trio bound to nucleotide-free Rac1. (a) The N-terminal DH (yellow) and PH (blue) domains of Trio are bound to nucleotide-depleted Rac1 (green with switch regions in red). Disordered regions are indicated with dotted lines. (b) Atomic details of the interface between Rac1 and the PH domain of Trio. Hydrogen bonds (2.6 – 4.0 Å) are indicated with dotted lines (c) A simulated annealing omit map (left) contoured at 1.0σ and a 2Fo-Fc map (right) contoured at 1.2σ generated using the final coordinates highlight the electron density at the interface between Rac1 and the PH domain. (d) The anisotropic motion of each atom is displayed as a thermal ellipse (left). An identical image without the thermal ellipses is shown as a reference (right). The interface between Rac1 and the PH domain of Trio, also depicted in Figure 2(b) and 2(c), is highlighted by the box.
Figure 3
Figure 3
Secondary structure of the DH/PH fragment of Trio. The crystal structure of the Trio fragment bound to Rac1 was used to define α-helices and β-strands according to nomenclature standardized for DH/PH cassettes. Secondary structure assignments were made using the program DSSP, coupled with visual assessment. Residues highlighted in red contribute to the interface between the PH domain of Trio and Rac1. Residues in gray are disordered and were not modeled.
Figure 4
Figure 4
The PH domains of Trio and Dbs interact similarly with their cognate GTPases. DH/PH fragments of Trio (a) and Dbs (b) in complex with their cognate GTPases have been superimposed upon equivalent, unbound fragments (gray) using the DH domains. (c and d) Lower panels highlight conserved interactions found in both GEF/GTPase complexes that require specific residues involving the PH domains. Color scheme is maintained from Figure 2.
Figure 5
Figure 5
Mutations that disrupt the interface between the PH domain of Trio and Rac1 diminish nucleotide exchange. Residues within, or supported by, the PH domain of Trio that form the interface with Rac1 were mutated and the exchange activities of the mutants were measured on both Rac1 (a) and RhoG (b). (c) Residues of Rac1 that interact directly with the PH domain of Trio were also analyzed. Exchange assays (n=2) were carried out as described in Methods. Exchange rates are reported as a percentage of the exchange rate of wild-type Trio (a and b) or as fold exchange over the intrinsic exchange rate of the respective mutant of Rac1 (c). Proteins (5 μg) were subjected to SDS-PAGE and stained with Coomassie Blue (insets) to verify purity and concentration. (d) Circular dichroism spectroscopy confirmed the proper folding of Trio DH/PH fragments.
Figure 6
Figure 6
Mutations within, or supported by, the PH domain of Trio that reduce GTPase activation in vitro also reduce the capacity of full-length Trio to induce neurite outgrowth in PC-12 cells. (a) Neurite outgrowth in transfected PC-12 cells was assessed as described in Methods (*; p-values < 0.05 in comparison to wild-type Trio using student's T-test). (b) Representative images of transfected PC-12 cells show both GFP fluorescence (left) and filamentouse actin stained with Alexa Fluor 546 phalloidin (right). All constructs were GFP-tagged at the N-terminus.
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References

    1. Dvorsky R, Ahmadian MR. Always look on the bright site of Rho: structural implications for a conserved intermolecular interface. EMBO Rep. 2004;5:1130–6. - PMC - PubMed
    1. Hoffman GR, Cerione RA. Signaling to the Rho GTPases: networking with the DH domain. FEBS Lett. 2002;513:85–91. - PubMed
    1. Etienne-Manneville S, Hall A. Rho GTPases in cell biology. Nature. 2002;420:629–35. - PubMed
    1. Schwartz MA, Shattil SJ. Signaling networks linking integrins and rho family GTPases. Trends Biochem Sci. 2000;25:388–91. - PubMed
    1. Sahai E, Marshall CJ. RHO-GTPases and cancer. Nat Rev Cancer. 2002;2:133–42. - PubMed

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