The GTPase-activating protein Rap1GAP uses a catalytic asparagine

Abstract

Rap1 is a Ras-like guanine-nucleotide-binding protein (GNBP) that is involved in a variety of signal-transduction processes1,2. It regulates integrin-mediated cell adhesion and might activate extracellular signal-regulated kinase. Like other Ras-like GNBPs, Rap1 is regulated by guanine-nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs). These GAPs increase the slow intrinsic GTPase reaction of Ras-like GNBPs by many orders of magnitude and allow tight regulation of signalling. The activation mechanism involves stabilization of the catalytic glutamine of the GNBP and, in most cases, the insertion of a catalytic arginine of GAP into the active site3. Rap1 is a close homologue of Ras but does not possess the catalytic glutamine essential for GTP hydrolysis in all other Ras-like and Gα proteins. Furthermore, RapGAPs are not related to other GAPs and apparently do not use a catalytic arginine residue4. Here we present the crystal structure of the catalytic domain of the Rap1-specific Rap1GAP at 2.9 Å. By mutational analysis, fluorescence titration and stopped-flow kinetic assay, we demonstrate that Rap1GAP provides a catalytic asparagine to stimulate GTP hydrolysis. Implications for the disease tuberous sclerosis are discussed.

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Figure 1: The structure of Rap1GAP.
Figure 2: Mapping the active site.
Figure 3: Mechanistic analysis of Rap1GAP catalysis.
Figure 4: Implications for tuberin.

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Acknowledgements

We thank A. Krämer for the gift of Rap1-Aedans·GTP. We thank I. Schlichting, W. Blankenfeldt, A. Scheidig, A. Rak, E. Wolf and O. Yildiz for data collection and crystallographic advice and the ESRF beam staff of beamline ID14-1 in Grenoble for support. O.D. thanks the Boehringer Ingelheim Fonds and P.P.C. the International Max-Planck Research School for support.

Author information

Correspondence to Alfred Wittinghofer.

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The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Figure 1

Sequence alignment of the Rap1GAP family and Tuberin. (PDF 36 kb)

Supplementary Figure 2

Superimposition of Rap1GAP and Ras. (JPG 48 kb)

Supplementary Figure 3

Ramachandran plot of the Rap1GAP model. (PDF 34 kb)

Supplementary Figure Legends (DOC 38 kb)

Supplementary Table 1

Phasing statistics of the Rap1GAP model. (PDF 7 kb)

Supplementary Table 2

Refinement statistics of the Rap1GAP model. (PDF 16 kb)

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