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Cited by 127Protein Kinase Regulation and GTPase SignalingCellular transport and secretionReceptor Mechanisms and Signaling

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The Rap–RapGAP complex: GTP hydrolysis without catalytic glutamine and arginine residues

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Scrima, Andrea, Thomas, Christoph, Deaconescu, Delia, Wittinghofer, Alfred·2008·The EMBO Journal
DOI 10.1038/emboj.2008.30PMC PMC2265111
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Abstract

The GTP-binding protein Rap1 regulates integrin-mediated and other cell adhesion processes. Unlike most other Ras-related proteins, it contains a threonine in switch II instead of a glutamine (Gln61 in Ras), a residue crucial for the GTPase reaction of most G proteins. Furthermore, unlike most other GTPase-activating proteins (GAPs) for small G proteins, which supply a catalytically important Arg-finger, no arginine residue of RapGAP makes a significant contribution to the GTPase reaction of Rap1. For a detailed understanding of the reaction mechanism, we have solved the structure of Rap1 in complex with Rap1GAP. It shows that the Thr61 of Rap is away from the active site and that an invariant asparagine of RapGAPs, the Asn-thumb, takes over the role of the cis-glutamine of Ras, Rho or Ran. The structure and biochemical data allow to further explain the mechanism and to define the important role of a conserved tyrosine. The structure and biochemical data furthermore show that the RapGAP homologous region of the tumour suppressor Tuberin is sufficient for catalysis on Rheb.

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Figures

Figure 1 Rap–Rap1GAP complex and interface analysis. (A) Ribbon representation of the Rap–GDP·BeF3−-Rap1GAP complex with Rap1B in cyan and Rap1GAP in green (catalytic domain green; dimerisation domain olive green). The catalytic helix containing the Asn-thumb (Asn290) is shown in magenta, GDP-BeF3− as ball-and-stick. (B) Schematic representation of interacting residues. Interactions shown in detail in (C–E) are depicted with a dashed line. (C–E) Structural details of interactions between Rap1B and Rap1GAP, with colours as in (A). (F) HPLC-based analysis of the Rap-stimulated GTPase reaction, with 200 μM wt and mutant Rap and 100 nM Rap1GAP. (G) Stopped-flow analysis of the interaction between 2 μM Aedans-labelled wt and mutant Rap and 50 μM Rap1GAP; reaction was followed by monitoring fluorescence through a 408 nm cutoff filter. Wt and mutant Rap contain the A86C mutation, which has been shown to behave as wild type, as described earlier (Kraemer et al, 2002; Chakrabarti et al, 2007).
Figure 1 Rap–Rap1GAP complex and interface analysis. (A) Ribbon representation of the Rap–GDP·BeF3−-Rap1GAP complex with Rap1B in cyan and Rap1GAP in green (catalytic domain green; dimerisation domain olive green). The catalytic helix containing the Asn-thumb (Asn290) is shown in magenta, GDP-BeF3− as ball-and-stick. (B) Schematic representation of interacting residues. Interactions shown in detail in (C–E) are depicted with a dashed line. (C–E) Structural details of interactions between Rap1B and Rap1GAP, with colours as in (A). (F) HPLC-based analysis of the Rap-stimulated GTPase reaction, with 200 μM wt and mutant Rap and 100 nM Rap1GAP. (G) Stopped-flow analysis of the interaction between 2 μM Aedans-labelled wt and mutant Rap and 50 μM Rap1GAP; reaction was followed by monitoring fluorescence through a 408 nm cutoff filter. Wt and mutant Rap contain the A86C mutation, which has been shown to behave as wild type, as described earlier (Kraemer et al, 2002; Chakrabarti et al, 2007).CC BY-NC-ND · PubMed Central
Figure 2 The active site. (A) Active site of Rap–Rap1GAP, shown as superimposition with Ras, Ran and Rho in complex with their cognate GAPs. Asn290 in Rap1GAP occupies the position of the catalytic Gln in Ras (Gln61), Ran (Gln69) and Rho (Gln63). The G12 position in Rap1B is marked with a sphere. (B) Superimposition of uncomplexed Rap (yellow) and the Rap–Rap1GAP (cyan/green) complex. Interaction of Gln63 and Phe64 with residues on Rap1GAP (green) forces switch II into an alternative conformation (arrows) to release blockade by Thr61 thereby allowing Asn290 to enter the active site. (C, D) Surface representation of uncomplexed Rap (C) and Rap in complex with Rap1GAP (D). The switch II residues T61 (red) and Q63/F64 (green) undergo a drastic conformational change upon complex formation with Rap1GAP to allow access for the Asn-thumb to the active site.
Figure 2 The active site. (A) Active site of Rap–Rap1GAP, shown as superimposition with Ras, Ran and Rho in complex with their cognate GAPs. Asn290 in Rap1GAP occupies the position of the catalytic Gln in Ras (Gln61), Ran (Gln69) and Rho (Gln63). The G12 position in Rap1B is marked with a sphere. (B) Superimposition of uncomplexed Rap (yellow) and the Rap–Rap1GAP (cyan/green) complex. Interaction of Gln63 and Phe64 with residues on Rap1GAP (green) forces switch II into an alternative conformation (arrows) to release blockade by Thr61 thereby allowing Asn290 to enter the active site. (C, D) Surface representation of uncomplexed Rap (C) and Rap in complex with Rap1GAP (D). The switch II residues T61 (red) and Q63/F64 (green) undergo a drastic conformational change upon complex formation with Rap1GAP to allow access for the Asn-thumb to the active site.CC BY-NC-ND · PubMed Central
Figure 3 Role of Tyr32. (A) Superimposition of active sites from various structures as indicated, with an emphasis on the conformation of Tyr32. Systems using an Arg-finger (RasGAP/RhoGAP) show Tyr32 in a more open versus a more closed conformation for Ran and Rap. (B) Surface representation of uncomplexed Rap/Ras or in complex with their respective GAPs, with residue Tyr32 labelled in yellow. The Rap or Ras structures are shown in slightly different orientations. Catalytic elements from the GAP, the catalytic helix with the Asn-thumb and the Arg-finger (magenta and brown, respectively) are shown as ribbon. (C) HPLC-based analysis of the GTPase stimulation by Rap1GAP for Rap wt and Y32-mutants (described in Figure 1F). (D) GTPase stimulation of Rap wt and Y32 mutants analysed by radioactive charcoal assay. Rap (10 μM) and Rap1GAP (50 nM) were incubated as described before (Kupzig et al, 2006). The concentration of released 32P-labelled Pi (correscponding to hydrolysed GTP) was plotted against the reaction time and initial rates were determined by linear regression fitting.
Figure 3 Role of Tyr32. (A) Superimposition of active sites from various structures as indicated, with an emphasis on the conformation of Tyr32. Systems using an Arg-finger (RasGAP/RhoGAP) show Tyr32 in a more open versus a more closed conformation for Ran and Rap. (B) Surface representation of uncomplexed Rap/Ras or in complex with their respective GAPs, with residue Tyr32 labelled in yellow. The Rap or Ras structures are shown in slightly different orientations. Catalytic elements from the GAP, the catalytic helix with the Asn-thumb and the Arg-finger (magenta and brown, respectively) are shown as ribbon. (C) HPLC-based analysis of the GTPase stimulation by Rap1GAP for Rap wt and Y32-mutants (described in Figure 1F). (D) GTPase stimulation of Rap wt and Y32 mutants analysed by radioactive charcoal assay. Rap (10 μM) and Rap1GAP (50 nM) were incubated as described before (Kupzig et al, 2006). The concentration of released 32P-labelled Pi (correscponding to hydrolysed GTP) was plotted against the reaction time and initial rates were determined by linear regression fitting.CC BY-NC-ND · PubMed Central
Figure 4 Stimulation of Rheb GTP hydrolysis by Tuberin. HPLC-based analysis of intrinsic and Tuberin-stimulated GTPase of Rheb, with two different constructs of the catalytic domain of Tuberin, using 80 μM Rheb and 100 μM Tuberin (Tuberinlong: 1532–1760; Tuberinshort: 1538–1729).
Figure 4 Stimulation of Rheb GTP hydrolysis by Tuberin. HPLC-based analysis of intrinsic and Tuberin-stimulated GTPase of Rheb, with two different constructs of the catalytic domain of Tuberin, using 80 μM Rheb and 100 μM Tuberin (Tuberinlong: 1532–1760; Tuberinshort: 1538–1729).CC BY-NC-ND · PubMed Central

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Metadata

Authors
Scrima, A., Thomas, C., Deaconescu, D., & Wittinghofer, A.
Affiliations
  • Max Planck Institute of Molecular Physiology (DE)
  • Max Planck Society (DE)
Corresponding author
Wittinghofer, Alfred
ORCID
Venue
The EMBO Journal
Publisher
Springer Science and Business Media LLC
Published
2008-02-28
Volume
27
Issue
7
Pages
1145-1153
ISSN
  • 0261-4189 (ISSN-L)
  • 1460-2075
DOI
10.1038/emboj.2008.30
PMC
PMC2265111
OpenAlex
W2104235973
Cited by
127
References
36
Citations by year
2026: 3 · 2025: 6 · 2024: 4 · 2023: 5 · 2022: 3 · 2021: 7 · 2020: 6 · 2019: 2 · 2018: 8 · 2017: 6 · 2016: 8 · 2015: 4 · 2014: 8 · 2013: 8 · 2012: 19
FWCI
4.11
Type
journal article
Language
en
Open access
Licence
Locations
  • The EMBO Journal (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • DORA PSI (Paul Scherrer Institute) (submittedVersion, cc-by)
  • Max Planck Institute for Plasma Physics (submittedVersion)
  • MPG.PuRe (Max Planck Society) (submittedVersion)
Funders
Paul Scherrer Institut
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Receptor Mechanisms and Signaling
Keywords
Rap1, Rap1GAP, GTPase-activating protein, arginine finger, integrin-mediated adhesion, tuberin, Rheb
MeSH terms
Tuberous Sclerosis Complex 2 Protein, Amino Acid Sequence, Arginine, Binding Sites, Catalysis, Glutamine, Guanosine Triphosphate, Hydrolysis, Models, Molecular, Molecular Sequence Data, Protein Binding, Protein Structure, Secondary, Crystallography, X-Ray, GTPase-Activating Proteins, rap1 GTP-Binding Proteins, Tumor Suppressor Proteins, Mutant Proteins

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