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, 9 (4), 350-5

The C2 Domain of SynGAP Is Essential for Stimulation of the Rap GTPase Reaction

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The C2 Domain of SynGAP Is Essential for Stimulation of the Rap GTPase Reaction

Vladimir Pena et al. EMBO Rep.

Abstract

The brain-specific synaptic guanosine triphosphatase (GTPase)-activating protein (SynGAP) is important in synaptic plasticity. It shows dual specificity for the small guanine nucleotide-binding proteins Rap and Ras. Here, we show that RapGAP activity of SynGAP requires its C2 domain. In contrast to the isolated GAP domain, which does not show any detectable RapGAP activity, a fragment comprising the C2 and GAP domains (C2-GAP) stimulates the intrinsic GTPase reaction of Rap by approximately 1 x 10(4). The C2-GAP crystal structure, complemented by modelling and biochemical analyses, favours a concerted movement of the C2 domain towards the switch II region of Rap to assist in GTPase stimulation. Our data support a catalytic mechanism similar to that of canonical RasGAPs and distinct from the canonical RapGAPs. SynGAP presents the first example, to our knowledge, of a GAP that uses a second domain for catalytic activity, thus pointing to a new function of C2 domains.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Structural overview and comparative kinetic analysis of synaptic guanosine triphosphatase-activating proteins. (A) Schematic representation of the SynGAP fragments used for biochemical and structural analysis. (B) Single-turnover measurements for the reaction of 0.1 μM Rap•tamraGTP and different SynGAP proteins at 5 μM concentration. (C) Ribbon diagram showing the overall structure of C2–GAP. The GAP region corresponding to the p120GAP domain responsible for catalysis (GAPc) with the helices presenting the Ras-binding groove highlighted in light yellow, the GAP region dispensable for catalysis (GAPex) and the C2 domain portions are shown in red, magenta and dark yellow, respectively. The C2 domain body in grey was modelled using Modeller and superposition onto the well-defined β-sheet near the domain interface. (D) Stereo view of the C2–GAP interface. The backbone (Cα trace) of the C2 domain is shown in yellow and that of the GAP domain in red. The composite omit map is contoured at 1.1σ. PH, pleckstrin homology; SynGAP, synaptic guanosine triphosphatase-activating protein; tamraGTP, 2′(3′)-O-(N-ethylcarbamoyl-(5″-carboxytetramethylrhodamine) amide)-GTP.
Figure 2
Figure 2
Structural regions crucial for the C2–GAP-stimulated GTPase of Rap. (A) Ribbon representation of the catalytic interface between SynGAP and Rap, as modelled by aligning Rap1 and C2–GAP based on the Ras–RasGAP complex (see panel C; Scheffzek et al, 1997). GAPc is shown in red, GAPex in magenta, the finger loop in yellow and Rap in grey. Residues that are thought to be important in catalysis are shown as sticks. (B) Single-turnover measurements of 0.1 μM Rap•tamraGTP and 5 μM SynGAP proteins as indicated. The observed rate constants determined from single exponential fitting are 0.28 s−1 for C2–GAP wild type, 0.023 s−1 for C2–GAP (N472T), 0.0031 s−1 for C2–GAP (R470K) and 0.0002 s−1 for C2–GAP (R470P). The intrinsic GTPase reaction rate is 0.0001 s−1. Data are the average of at least three individual measurements. (C) Hypothetical complex of C2–GAP with Rap based on a structural alignment of GAPc and Rap with the corresponding portions of the Ras–RasGAP complex (Scheffzek et al, 1997). The two monomers from the asymmetric unit were superimposed to show the shift of the GAPex (coil) and C2 domain (β-strands) towards the presumed Rap–GAPc interface. The body of the C2 domain is included as an ellipsoid to visualize the potential conformational shift. GAPc is depicted as a dark pink surface. GTPase, guanosine triphosphatase; sw II, switch II region; SynGAP, synaptic guanosine triphosphatase-activating protein; tamraGTP, 2′(3′)-O-(N-ethylcarbamoyl-(5″-carboxytetramethylrhodamine) amide)-GTP.

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