On the mechanism of autoinhibition of the RhoA-specific nucleotide exchange factor PDZRhoGEF

Here we show that the autoinhibition of PRG is caused largely by an interaction of a short negatively charged sequence motif, immediately upstream of the DH-domain and including residues Asp706, Glu708, Glu710 and Asp712, with a patch on the catalytic surface of the DH-domain including Arg867 and Arg868. In the absence of both PDZ and RGSL domains, the DH-PH tandem with additional 21 residues upstream, is 50% autoinhibited. However, within the full-length protein, the PDZ and/or RGSL domains significantly restore autoinhibition.Our results suggest a mechanism for autoinhibition of RGSL family of GEFs, in which the RGSL domain and a unique sequence motif upstream of the DH domain, act cooperatively to reduce the ability of the DH domain to bind the nucleotide free RhoA. The activation mechanism is likely to involve two independent steps, i.e. displacement of the RGSL domain and conformational change involving the autoinhibitory sequence motif containing several negatively charged residues.Rho (Ras-homology) cytosolic GTPases function as molecular switches that, in the GTP-bound form, interact with a multitude of effectors that exert control over cytoskeletal elements, gene transcription, and other biological phenomena [1-3]. Spatial and temporal control over these GTPases is exercised by GEFs (guanine nucleotide exchange factors), which load up GTP and activate cognate GTPases, and by GAPs (GTPase activating proteins) which are required by the GTPase for efficient hydrolysis of GTP to GDP [4,5]. Most of Rho GEFs belong to the Dbl-homology family of large, multidomain proteins [6]. There are approximately 70 of these proteins in the human proteome, some highly specific and some activating indiscriminately two or more different Rho GTPases [5,7]. The catalytic step is executed by the Dbl-homology (DH) domain, often assisted by a pleckstrin-homology (PH) domain, which is invariably located immediately downstream of the DH domain [6,7]. The DH domain, either alone or syn