Domain topology of human Rasal

Jorge Cuellar 1 , José María Valpuesta 1 , 2 , Alfred Wittinghofer 3  and Begoña Sot 1 , 2 , 4
  • 1 Department of Macromolecular Structures, Centro Nacional de Biotecnología (CNB-CSIC), Madrid, Spain
  • 2 Unidad Asociada de Nanobiotecnología (CNB-CSIC e IMDEA Nanociencia), Madrid, Spain
  • 3 Department of Structural Biology, Max-Planck-Institute for Molecular Physiology, Dortmund, Germany
  • 4 IMDEA-Nanociencia, Faraday 9, Campus Universitario de Cantoblanco, 28048 Madrid, Spain
Jorge Cuellar
  • Department of Macromolecular Structures, Centro Nacional de Biotecnología (CNB-CSIC), Madrid, Spain
  • Search for other articles:
  • degruyter.comGoogle Scholar
, José María Valpuesta
  • Department of Macromolecular Structures, Centro Nacional de Biotecnología (CNB-CSIC), Madrid, Spain
  • Unidad Asociada de Nanobiotecnología (CNB-CSIC e IMDEA Nanociencia), Madrid, Spain
  • Search for other articles:
  • degruyter.comGoogle Scholar
, Alfred Wittinghofer
  • Department of Structural Biology, Max-Planck-Institute for Molecular Physiology, Dortmund, Germany
  • Search for other articles:
  • degruyter.comGoogle Scholar
and Begoña Sot
  • Corresponding author
  • Department of Macromolecular Structures, Centro Nacional de Biotecnología (CNB-CSIC), Madrid, Spain
  • Unidad Asociada de Nanobiotecnología (CNB-CSIC e IMDEA Nanociencia), Madrid, Spain
  • IMDEA-Nanociencia, Faraday 9, Campus Universitario de Cantoblanco, 28048 Madrid, Spain
  • Email
  • Search for other articles:
  • degruyter.comGoogle Scholar


Rasal is a modular multi-domain protein of the GTPase-activating protein 1 (GAP1) family; its four known members, GAP1m, Rasal, GAP1IP4BP and Capri, have a Ras GTPase-activating domain (RasGAP). This domain supports the intrinsically slow GTPase activity of Ras by actively participating in the catalytic reaction. In the case of Rasal, GAP1IP4BP and Capri, their remaining domains are responsible for converting the RasGAP domains into dual Ras- and Rap-GAPs, via an incompletely understood mechanism. Although Rap proteins are small GTPase homologues of Ras, their catalytic residues are distinct, which reinforces the importance of determining the structure of full-length GAP1 family proteins. To date, these proteins have not been crystallized, and their size is not adequate for nuclear magnetic resonance (NMR) or for high-resolution cryo-electron microscopy (cryoEM). Here we present the low resolution structure of full-length Rasal, obtained by negative staining electron microscopy, which allows us to propose a model of its domain topology. These results help to understand the role of the different domains in controlling the dual GAP activity of GAP1 family proteins.

    • Supplementary material
  • Alvira, S., Cuellar, J., Rohl, A., Yamamoto, S., Itoh, H., Alfonso, C., Rivas, G., Buchner, J., and Valpuesta, J.M. (2014). Structural characterization of the substrate transfer mechanism in Hsp70/Hsp90 folding machinery mediated by Hop. Nat. Commun. 5, 5484.

  • Beckmann, R., Bubeck, D., Grassucci, R., Penczek, P., Verschoor, A., Blobel, G., and Frank, J. (1997). Alignment of conduits for the nascent polypeptide chain in the ribosome-Sec61 complex. Science 278, 2123–2126.

  • Dasgupta, B. and Gutmann, D.H. (2003). Neurofibromatosis 1: closing the GAP between mice and men. Curr. Opin. Genet. Dev. 13, 20–27.

  • Daumke, O., Weyand, M., Chakrabarti, P.P., Vetter, I.R., and Wittinghofer, A. (2004). The GTPase-activating protein Rap1GAP uses a catalytic asparagine. Nature 429, 197–201.

  • Downward, J. (2003). Targeting RAS signalling pathways in cancer therapy. Nat. Rev. Cancer 3, 11–22.

  • Jin, H., Wang, X., Ying, J., Wong, A.H., Cui, Y., Srivastava, G., Shen, Z.Y., Li, E.M., Zhang, Q., Jin, J., et al. (2007). Epigenetic silencing of a Ca(2+)-regulated Ras GTPase-activating protein RASAL defines a new mechanism of Ras activation in human cancers. Proc. Natl. Acad. Sci. USA 104, 12353–12358.

  • Kim, J.H., Lee, H.K., Takamiya, K., and Huganir, R.L. (2003). The role of synaptic GTPase-activating protein in neuronal development and synaptic plasticity. J. Neurosci. 23, 1119–1124.

  • Kupzig, S., Bouyoucef-Cherchalli, D., Yarwood, S., Sessions, R., and Cullen, P.J. (2009). The ability of GAP1IP4BP to function as a Rap1 GTPase-activating protein (GAP) requires its Ras GAP-related domain and an arginine finger rather than an asparagine thumb. Mol. Cell Biol. 29, 3929–3940.

  • Kupzig, S., Deaconescu, D., Bouyoucef, D., Walker, S.A., Liu, Q., Polte, C.L., Daumke, O., Ishizaki, T., Lockyer, P.J., Wittinghofer, A. et al. (2006). GAP1 family members constitute bifunctional Ras and Rap GTPase-activating proteins. J. Biol. Chem. 281, 9891–9900.

  • Lander, G.C., Estrin, E., Matyskiela, M.E., Bashore, C., Nogales, E., and Martin, A. (2012). Complete subunit architecture of the proteasome regulatory particle. Nature 482, 186–191.

  • Liu, Q., Walker, S.A., Gao, D., Taylor, J.A., Dai, Y.F., Arkell, R.S., Bootman, M.D., Roderick, H.L., Cullen, P.J., and Lockyer, P.J. (2005). CAPRI and RASAL impose different modes of information processing on Ras due to contrasting temporal filtering of Ca2+. J. Cell Biol. 170, 183–190.

  • Ludtke, S.J., Baldwin, P.R., and Chiu, W. (1999). EMAN: semiautomated software for high-resolution single-particle reconstructions. J. Struct. Biol. 128, 82–97.

  • Marabini, R., Masegosa, I.M., San Martin, M.C., Marco, S., Fernandez, J.J., de la Fraga, L.G., Vaquerizo, C., and Carazo, J.M. (1996). Xmipp: an image processing package for electron microscopy. J. Struct. Biol. 116, 237–240.

  • Mindell, J.A. and Grigorieff, N. (2003). Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347.

  • Ohta, M., Seto, M., Ijichi, H., Miyabayashi, K., Kudo, Y., Mohri, D., Asaoka, Y., Tada, M., Tanaka, Y., Ikenoue, T., et al. (2009). Decreased expression of the RAS-GTPase activating protein RASAL1 is associated with colorectal tumor progression. Gastroenterology 136, 206–216.

  • Pamonsinlapatham, P., Hadj-Slimane, R., Lepelletier, Y., Allain, B., Toccafondi, M., Garbay, C., and Raynaud, F. (2009). p120-Ras GTPase activating protein (RasGAP): a multi-interacting protein in downstream signaling. Biochimie 91, 320–328.

  • Pena, V., Hothorn, M., Eberth, A., Kaschau, N., Parret, A., Gremer, L., Bonneau, F., Ahmadian, M.R., and Scheffzek, K. (2008). The C2 domain of SynGAP is essential for stimulation of the Rap GTPase reaction. EMBO Rep. 9, 350–355.

  • Penczek, P.A. (2002). Three-dimensional spectral signal-to-noise ratio for a class of reconstruction algorithms. J. Struct. Biol. 138, 34–46.

  • Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C., and Ferrin, T.E. (2004). UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612.

  • Querol-Audi, J., Sun, C., Vogan, J.M., Smith, M.D., Gu, Y., Cate, J.H., and Nogales, E. (2013). Architecture of human translation initiation factor 3. Structure 21, 920–928.

  • Raaijmakers, J.H. and Bos, J.L. (2009). Specificity in Ras and Rap signaling. J. Biol. Chem. 284, 10995–10999.

  • Scheffzek, K., Ahmadian, M.R., Kabsch, W., Wiesmuller, L., Lautwein, A., Schmitz, F., and Wittinghofer, A. (1997). The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants. Science 277, 333–338.

  • Scheffzek, K., Ahmadian, M.R., Wiesmuller, L., Kabsch, W., Stege, P., Schmitz, F., and Wittinghofer, A. (1998). Structural analysis of the GAP-related domain from neurofibromin and its implications. EMBO J. 17, 4313–4327.

  • Scheres, S.H. (2010). Classification of structural heterogeneity by maximum-likelihood methods. Methods Enzymol. 482, 295–320.

  • Scheres, S.H., Valle, M., Nunez, R., Sorzano, C.O., Marabini, R., Herman, G.T., and Carazo, J.M. (2005). Maximum-likelihood multi-reference refinement for electron microscopy images. J. Mol. Biol. 348, 139–149.

  • Scrima, A., Thomas, C., Deaconescu, D., and Wittinghofer, A. (2008). The Rap-RapGAP complex: GTP hydrolysis without catalytic glutamine and arginine residues. EMBO J. 27, 1145–1153.

  • Sorzano, C.O., Bilbao-Castro, J.R., Shkolnisky, Y., Alcorlo, M., Melero, R., Caffarena-Fernandez, G., Li, M., Xu, G., Marabini, R., and Carazo, J.M. (2010). A clustering approach to multireference alignment of single-particle projections in electron microscopy. J. Struct. Biol. 171, 197–206.

  • Sot, B., Behrmann, E., Raunser, S., and Wittinghofer, A. (2013). Ras GTPase activating (RasGAP) activity of the dual specificity GAP protein Rasal requires colocalization and C2 domain binding to lipid membranes. Proc. Natl. Acad. Sci. USA 110, 111–116.

  • Sot, B., Kotting, C., Deaconescu, D., Suveyzdis, Y., Gerwert, K., and Wittinghofer, A. (2010). Unravelling the mechanism of dual-specificity GAPs. EMBO J. 29, 1205–1214.

  • Ukleja, M., Cuellar, J., Siwaszek, A., Kasprzak, J.M., Czarnocki-Cieciura, M., Bujnicki, J.M., Dziembowski, A., and Valpuesta, J.M. (2016). The architecture of the Schizosaccharomyces pombe CCR4-NOT complex. Nat. Commun. 7, 10433.

  • van Heel, M. and Schatz, M. (2005). Fourier shell correlation threshold criteria. J. Struct. Biol. 151, 250–262.

  • Walker, S.A., Kupzig, S., Bouyoucef, D., Davies, L.C., Tsuboi, T., Bivona, T.G., Cozier, G.E., Lockyer, P.J., Buckler, A., Rutter, G.A., et al. (2004). Identification of a Ras GTPase-activating protein regulated by receptor-mediated Ca2+ oscillations. EMBO J. 23, 1749–1760.

  • Walkup, W. G., Washburn, L., Sweredoski, M.J., Carlisle, H.J., Graham, R.L., Hess, S., and Kennedy, M.B. (2015). Phosphorylation of synaptic GTPase-activating protein (synGAP) by Ca2+/calmodulin-dependent protein kinase II (CaMKII) and cyclin-dependent kinase 5 (CDK5) alters the ratio of its GAP activity toward Ras and Rap GTPases. J. Biol. Chem. 290, 4908–4927.

  • Wang, Y., Pascoe, H.G., Brautigam, C.A., He, H., and Zhang, X. (2013). Structural basis for activation and non-canonical catalysis of the Rap GTPase activating protein domain of plexin. Elife 2, e01279.

  • Waterhouse, A.M., Procter, J.B., Martin, D.M., Clamp, M., and Barton, G.J. (2009). Jalview Version 2–a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189–1191.

  • Yang, J. and Zhang, Y. (2015). Protein structure and function prediction using I-TASSER. Curr. Protoc. Bioinformatics 52, 5.8.1–5.815.

Purchase article
Get instant unlimited access to the article.
Log in
Already have access? Please log in.

Log in with your institution

Journal + Issues

Biological Chemistry keeps you up-to-date with the latest advances in the molecular life sciences. The journal publishes Research Articles, Short Communications, Reviews and Minireviews. Areas include: general biochemistry/pathobiochemistry, structural biology, molecular and cellular biology, genetics and epigenetics, virology, molecular medicine, plant molecular biology/biochemistry and novel experimental methodologies.