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. 2009 May 21:9:36.
doi: 10.1186/1472-6807-9-36.

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

Meiying Zheng  1 , Tomasz Cierpicki, Ko Momotani, Mykhaylo V Artamonov, Urszula Derewenda, John H Bushweller, Avril V Somlyo, Zygmunt S Derewenda
Affiliations

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

Meiying Zheng et al. BMC Struct Biol. .

Abstract

Background: The Dbl-family of guanine nucleotide exchange factors (GEFs) activate the cytosolic GTPases of the Rho family by enhancing the rate of exchange of GTP for GDP on the cognate GTPase. This catalytic activity resides in the DH (Dbl-homology) domain, but typically GEFs are multidomain proteins containing other modules. It is believed that GEFs are autoinhibited in the cytosol due to supramodular architecture, and become activated in diverse signaling pathways through conformational change and exposure of the DH domain, as the protein is translocated to the membrane. A small family of RhoA-specific GEFs, containing the RGSL (regulators of G-protein signaling-like) domain, act as effectors of select GPCRs via Galpha12/13, although the molecular mechanism by which this pathway operates is not known. These GEFs include p115, LARG and PDZRhoGEF (PRG).

Results: 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.

Conclusion: 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.

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Figures

Figure 1
Figure 1
Multidomain fragments and mutants of PRG generated in this study and their functional characterization; (A) diagrammatic representation of the expression vector used for all constructs; (B) The constructs used in this study, the kcat values and fold-enhancement of nucleotide exchange compared to uncatalyzed RhoA.
Figure 2
Figure 2
Representative results of the nucleotide exchange assay. Only half of experimental points are visualized in the graph. For other details refer to Materials and Methods.
Figure 3
Figure 3
The 800 MHz 1H-15N TROSY-HSQC spectrum of the 0.3 mM DH-PH tandem at 30°C; assignment is shown for two selected fragments of the spectrum.
Figure 4
Figure 4
The fragment containing residues 672–712, upstream of the DH-PH tandem, interacts with residues within the DH domain and with RhoA in the binary complex. (A) comparison of 900 MHz 1H-15N TROSY-HSQC spectra of DH-PH (blue) and PRG672–1081 (red) showing chemical shift changes within DH domain induced by the linker; only select fragments of the spectrum are shown for clarity (B) a graph showing the magnitude of linker induced chemical shift changes (ΔσHN [Hz]) within the DH domain (blue) compared to the surface area of DH residues buried upon formation of the complex with RhoA (red); ΔσHN [Hz] were calculated as a differences between chemical shifts for PRG672–1081and DH-PH; the surface buried upon complex formation was calculated using the crystal structure of DHPH-RhoA complex and the program MOLMOL [38]; (C) crystal structure of the DH-PH/RhoA complex (PDB code 1XCG), showing the residues within the DH domain with the largest amide chemical shift changes induced by the linker (>50 Hz, red; 25 – 50 Hz, orange); the hypothetical position of the linker, postulated on the basis of chemical shift perturbations, is shown in yellow; side chains of acidic residues within the linker are shown in space filling representation; RhoA is shown as blue ribbon; (D) comparison of 600 MHz 1H-15N TROSY-HSQC spectra of RhoA in complex with DH-PH (blue) and PRG672–1081 (red); several RhoA amides with chemical shifts perturbed by the presence of the linker are circled.
Figure 5
Figure 5
The impact of mutations in the linker region on its interaction with the DH domain and with RhoA. (A) comparison of 600 MHz 1H-15N TROSY-HSQC spectra of PRG672–1081 (black), the two mutants: PRG3R (red) and PRG4R (blue) and DH-PH (green); (B) plot of chemical shift differences (ΔσHN) within DH domain induced by the wild-type linker (black), PRG3R (red) and PRG4R mutants (blue); (C) comparison of 600 MHz 1H-15N TROSY-HSQC spectra of 2H,15N-RhoA in complex with wild-type PRG672–1081 (red) and PRG4R (black); several amides with the largest chemical shift perturbations are circled, peaks labeled with asterisks could not be identified in the spectrum of PRG4R; (D) surface charge distribution calculated for the crystal structure of DH-PH/RhoA complex; the putative position of the linker is shown in yellow; side chains of acidic residues within the linker are shown in space filling representation; RhoA is delineated by a green boundary for clarity.
Figure 6
Figure 6
Contractility assay. Wild type (wt) and 4R mutant of the core fragment of PRG were added to β-escin permeabilized pulmonary artery precontracted with pCa 6.7 solution containing 1 μM calmodulin and 2 μM GTP. Concomitant increase in force is consistent with activation of the RhoA pathway. The 4R mutant, 10–15 μM, induced significantly greater Ca2+-sensitized force than the wild type protein (p = 0.002; n = 11. The magnitudes of the responses are normalized to pCa 6.7-induced force developed before addition of the recombinant PRG variants.
Figure 7
Figure 7
Increase in the endogenous RhoA activation in NIH 3T3 cells monitored by the Rhotekin assay. Upper panel: Rhotekin pull-down of RhoA•GTP from cells ectopically expressing full-length wild type (wt) PRG, the isolated DH-PH tandem and the 3R and 4R mutants of the full-length form. The cells were under serum free conditions. Bottom panel: quantitative assessment of RhoA activation using a set of three independent experiments. The isolated DH-PH tandem is not included because the expression level was significantly higher than that of the three full-length PRG variants, and while the increased endogenous RhoA activity as measured by the Rhotekin assay (see Methods Section), demonstrating that these expressed constructs are biologically active in cells. Mutant PRG-RhoGEFS were FLAG tagged. Western blotting for FLAG indicated that the mutants were expressed at an equal level while the DH-PH domain was overexpressed (data not shown) accounting for the greater level of active RhoA.
Figure 8
Figure 8
A model of autoinhibition and activation of PDZRhoGEF. In the inhibited protein the linker between the RGSL and DH domains is sequestered by the two modules, and the DH domain is not accessible to RhoA. Interaction with the Gα subunit, and possibly direct interaction with relevant GPCRs via the PDZ domain, relieve this inhibition by first dislodging the RGSL domain and then the inhibitory portion of the linker.
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