The Structural Insights into the Working of the Multidomain, RhoA-specific Guanine Nucleotide Exchange Factor, PDZRhoGEF

PDZRhoGEF belongs to a small family of RhoA-specific nucleotide exchange factors, which mediate signaling through select G-protein coupled receptors (GPCRs) via G 13 , and activate RhoA by catalyzing the exchange of GDP to GTP. PDZRhoGEF is a multidomain protein containing PDZ, RGSL, DH and PH domains. It is autoinhibited in cytosol, and is believed to undergo a conformational rearrangement and translocation to the membrane for full activation, although the molecular details of the regulation mechanism are not clear. It has been shown recently, that the main autoregulatory elements of PDZRhoGEF – the autoinhibitory "activation box" and the "GEF switch", which is required for full activation, are located within the RGSL-DH linker, directly upstream of the catalytic DH domain and its RhoA-binding surface. Here, using a combination of biophysical and biochemical methods, I show that the mechanism of PDZRhoGEF regulation is more complex, and may involve an additional autoinhibitory element in the form of a molten globule region within the linker between RGSL and DH domains. I propose a novel, two-tier model of autoinhibition, where the „activation box‟ and the molten globule region act synergistically to impair the ability of RhoA to bind to the catalytic DH-PH tandem. The molten globule region and the "activation box" become less ordered in the PDZRhoGEF /RhoA complex, and dissociate from the RhoA binding site, which may constitute a critical step leading to PDZRhoGEF activation. All RGS- GEFs seem to share these regulatory features. It is therefore possible, that the regulation mechanism proposed in this study might be universal for this family of GEFs. The results ii presented in this thesis extend the current knowledge about regulation of PDZRhoGEF, and may lead to the full understanding of the molecular basis of this complex process. Jakub A.

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