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, 14 (12), e0226113
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Crystal Structures of p120RasGAP N-terminal SH2 Domain in Its Apo Form and in Complex With a p190RhoGAP Phosphotyrosine Peptide

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Crystal Structures of p120RasGAP N-terminal SH2 Domain in Its Apo Form and in Complex With a p190RhoGAP Phosphotyrosine Peptide

Rachel Jaber Chehayeb et al. PLoS One.

Erratum in

Abstract

The Rho and Ras pathways play vital roles in cell growth, division and motility. Cross-talk between the pathways amplifies their roles in cell proliferation and motility and its dysregulation is involved in disease pathogenesis. One important interaction for cross-talk occurs between p120RasGAP (RASA1), a GTPase activating protein (GAP) for Ras, and p190RhoGAP (p190RhoGAP-A, ARHGAP35), a GAP for Rho. The binding of these proteins is primarily mediated by two SH2 domains within p120RasGAP engaging phosphorylated tyrosines of p190RhoGAP, of which the best studied is pTyr-1105. To better understand the interaction between p120RasGAP and p190RhoGAP, we determined the 1.75 Å X-ray crystal structure of the N-terminal SH2 domain of p120RasGAP in the unliganded form, and its 1.6 Å co-crystal structure in complex with a synthesized phosphotyrosine peptide, EEENI(p-Tyr)SVPHDST, corresponding to residues 1100-1112 of p190RhoGAP. We find that the N-terminal SH2 domain of p120RhoGAP has the characteristic SH2 fold encompassing a central beta-sheet flanked by two alpha-helices, and that peptide binding stabilizes specific conformations of the βE-βF loop and arginine residues R212 and R231. Site-directed mutagenesis and native gel shifts confirm phosphotyrosine binding through the conserved FLVR motif arginine residue R207, and isothermal titration calorimetry finds a dissociation constant of 0.3 ± 0.1 μM between the phosphopeptide and SH2 domain. These results demonstrate that the major interaction between two important GAP proteins, p120RasGAP and p190RhoGAP, is mediated by a canonical SH2-pTyr interaction.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Structure of apo p120RasGAP SH2 domain and its complex with p190RhoGAP pTyr-1105 peptide.
A) Domain organizations of p190RhoGAP-A and p120RasGAP. The region that is co-crystallized is highlighted by a dashed box. Domains are indicated: FF, FF domain; SH2, Src-homology 2; SH3, Src-homology 3; PH, pleckstrin homology; C2, C2 domain. C-terminal residue number labeled. B) Overall structure of the N-terminal SH2 domain of p120RasGAP. Secondary structure elements are indicated. Locations of the pTyr binding site, and the specificity determining +3 site are indicated. C) Overall structure of the N-terminal SH2 domain of p120RasGAP in complex with a peptide corresponding to residues 1100–1112 of p120RasGAP. p120RasGAP is shown in cartoon format. p190RhoGAP is shown in stick format. pTyr-1105 is indicated. 2Fobs-Fcalc electron density for the p190RhoGAP peptide is shown at a contour level of 1σ in blue. Positions of peptide residues are indicated. D) Side view of C, highlighting positions and density of pTyr-1105 and Pro-1108.
Fig 2
Fig 2. Conformational sampling of the apo structures of p120RasGAP N-SH2.
A) Conformational differences between the poised (pink) and occluded (grey) states observed in the asymmetric unit of the crystal structure. B) Superposition of the poised and occluded states onto the complexed structure and indicates that the occluded conformation clashes with peptide residue His-1109.
Fig 3
Fig 3. Features of the p120RasGAP-p190RhoGAP co-crystal structure.
A) Close-up of the interactions between p190RhoGAP pTyr-1105 (yellow, stick) and p120RasGAP SH2 domain (blue, cartoon and stick). FLVR motif arginine is Arg-207. H-bonds are shown in orange. B) Cation-π stack between pTyr-1105, Arg-231 and Arg-212. C) Close-up of the +3 pocket. D) Surface electrostatics of p120RasGAP. p190RhoGAP shown in stick format.
Fig 4
Fig 4. Native PAGE for p120RasGAP N-SH2 and p190RhoGAP pTyr-1105 peptide.
Native PAGE illustrating the effect of increasing peptide concentrations on wild-type p120RasGAP N-SH2 (left) and R207A mutant p120RasGAP (right). Peptide:protein molar ratio indicated for each lane.
Fig 5
Fig 5. Isothermal titration calorimetry p190RhoGAP pTyr-1105 peptide titration into p120RasGAP N-SH2.
A) p190RhoGAP pTyr-1105 peptide at 78.5 μM titrated into p120RasGAP N-SH2 at 15 μM. B) p190RhoGAP pTyr-1105 peptide at 120 μM titrated into R207A p120RasGAP N-SH2 at 15 μM.

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