Insights into the Molecular Activation Mechanism of the RhoA-specific Guanine Nucleotide Exchange Factor, PDZRhoGEF

Dmitri I Svergun,Alexander V Shkumatov,Urszula Derewenda,Zygmunt S Derewenda,Jakub A Bielnicki,Avril V Somlyo

doi:10.1074/jbc.m111.270918

Dmitri I Svergun, Alexander V Shkumatov + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m111.270918

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Abstract

PDZRhoGEF (PRG) belongs to a small family of RhoA-specific nucleotide exchange factors that mediates signaling through select G-protein-coupled receptors via Gα(12/13) and activates RhoA by catalyzing the exchange of GDP to GTP. PRG is a multidomain protein composed of PDZ, regulators of G-protein signaling-like (RGSL), Dbl-homology (DH), and pleckstrin-homology (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 directly upstream of the catalytic DH domain and its RhoA binding surface, emphasizing the functional role of the RGSL-DH linker. Here, using a combination of biophysical and biochemical methods, we show that the mechanism of PRG regulation is yet 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. We 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 PRG-RhoA complex and dissociate from the RhoA-binding site, which may constitute a critical step leading to PRG activation.

Highlights

For the cell to respond to specific stimuli with RhoA activation, the GEFs are autoinhibited in their native state and assume an active form only in response to such stimulation
Downstream of this module there is a C-terminal domain of unknown structure and function that may mediate in vivo homo- and heterodimerization of the regulators of G-protein signaling-like (RGSL)-family GEFs [47, 48] but is not important for the catalytic activity
Our results show conclusively that the functional fragment of PRG encompassing the four globular domains (PDZ, RGSL, DH, and PH) does not have a simple supramodular architecture in which the domains would interact directly to form a globular moiety

Summary

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—Truncated fragments of human PRG (Fig. 1) were subcloned into pDEST15 vector (Invitrogen) with a GST tag followed by a tobacco etch virus cleavage site, target sequence, and a C-terminal noncleavable His tag [25]. For experiments involving free PRG fragments, fractions were pooled, concentrated, and re-applied on Superdex-200 gel filtration column (Amersham Biosciences) pre-equilibrated with buffer C to remove any aggregated protein. Hydrogen/deuterium isotopic exchange in the PRG 37–1081 fragment was initiated by mixing 10 ␮l of the protein solution (ϳ1.3 mg/ml) with 10 ␮l of deuterated buffer D (50 mM citrate, pH 6.0) at 23 °C. PRG constructs were reconstructed ab initio from the scattering data using DAMMIN [36] and DAMMIF [37] These programs represent the particle by an assembly of densely packed spheres and employ simulated annealing to construct a compact interconnected model fitting the experimental data to minimize the discrepancy, ͱ ͸ͫ ͬ ␹ ϭ. The structure was determined using the molecular replacement pipeline BALBES [45] and refined with PHENIX [46] to final R and Rfree values of 21.1 and 27.1%, respectively

RESULTS

Mr SLS

Dmax vp

Root mean square deviations

DISCUSSION