Skip to content
Licensed Unlicensed Requires Authentication Published by De Gruyter September 8, 2017

Domain topology of human Rasal

Jorge Cuellar, José María Valpuesta, Alfred Wittinghofer and Begoña Sot
From the journal Biological Chemistry


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.


We thank Dr. Mónica Chagoyen (CNB-CSIC) for building the Ct domain model and Unidad de Proteómica of Centro Nacional de Biotecnología (CNB-CSIC) for XL-MS experiments and Catherine Mark for editorial assistance. This work was supported by Spanish Ministry of Economy and Innovation grants BFU2016-75984 (to JMV) and grants RYC-2011-08746 and Bolsa de Investigación L’Oréal-UNESCO 2013 (to BS).


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, 5484.10.1038/ncomms6484Search in Google Scholar

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, 2123–2126.10.1142/9789813234864_0020Search in Google Scholar

Dasgupta, B. and Gutmann, D.H. (2003). Neurofibromatosis 1: closing the GAP between mice and men. Curr. Opin. Genet. Dev. 13, 20–27.10.1016/S0959-437X(02)00015-1Search in Google Scholar

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, 197–201.10.1038/nature02505Search in Google Scholar

Downward, J. (2003). Targeting RAS signalling pathways in cancer therapy. Nat. Rev. Cancer 3, 11–22.10.1038/nrc969Search in Google Scholar

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, 12353–12358.10.1073/pnas.0700153104Search in Google Scholar

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, 1119–1124.10.1523/JNEUROSCI.23-04-01119.2003Search in Google Scholar

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, 3929–3940.10.1128/MCB.00427-09Search in Google Scholar

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, 9891–9900.10.1074/jbc.M512802200Search in Google Scholar

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, 186–191.10.1038/nature10774Search in Google Scholar

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, 183–190.10.1083/jcb.200504167Search in Google Scholar

Ludtke, S.J., Baldwin, P.R., and Chiu, W. (1999). EMAN: semiautomated software for high-resolution single-particle reconstructions. J. Struct. Biol. 128, 82–97.10.1006/jsbi.1999.4174Search in Google Scholar

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, 237–240.10.1006/jsbi.1996.0036Search in Google Scholar

Mindell, J.A. and Grigorieff, N. (2003). Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347.10.1016/S1047-8477(03)00069-8Search in Google Scholar

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, 206–216.10.1053/j.gastro.2008.09.063Search in Google Scholar

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, 320–328.10.1016/j.biochi.2008.10.010Search in Google Scholar

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, 350–355.10.1038/embor.2008.20Search in Google Scholar

Penczek, P.A. (2002). Three-dimensional spectral signal-to-noise ratio for a class of reconstruction algorithms. J. Struct. Biol. 138, 34–46.10.1016/S1047-8477(02)00033-3Search in Google Scholar

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, 1605–1612.10.1002/jcc.20084Search in Google Scholar

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, 920–928.10.1016/j.str.2013.04.002Search in Google Scholar

Raaijmakers, J.H. and Bos, J.L. (2009). Specificity in Ras and Rap signaling. J. Biol. Chem. 284, 10995–10999.10.1074/jbc.R800061200Search in Google Scholar

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, 333–338.10.1126/science.277.5324.333Search in Google Scholar

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, 4313–4327.10.1093/emboj/17.15.4313Search in Google Scholar

Scheres, S.H. (2010). Classification of structural heterogeneity by maximum-likelihood methods. Methods Enzymol. 482, 295–320.10.1016/S0076-6879(10)82012-9Search in Google Scholar

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, 139–149.10.1016/j.jmb.2005.02.031Search in Google Scholar

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, 1145–1153.10.1038/emboj.2008.30Search in Google Scholar

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, 197–206.10.1016/j.jsb.2010.03.011Search in Google Scholar

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, 111–116.10.1073/pnas.1201658110Search in Google Scholar

Sot, B., Kotting, C., Deaconescu, D., Suveyzdis, Y., Gerwert, K., and Wittinghofer, A. (2010). Unravelling the mechanism of dual-specificity GAPs. EMBO J. 29, 1205–1214.10.1038/emboj.2010.20Search in Google Scholar

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, 10433.10.1038/ncomms10433Search in Google Scholar

van Heel, M. and Schatz, M. (2005). Fourier shell correlation threshold criteria. J. Struct. Biol. 151, 250–262.10.1016/j.jsb.2005.05.009Search in Google Scholar

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, 1749–1760.10.1038/sj.emboj.7600197Search in Google Scholar

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, 4908–4927.10.1074/jbc.M114.614420Search in Google Scholar

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, e01279.10.7554/eLife.01279.020Search in Google Scholar

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, 1189–1191.10.1093/bioinformatics/btp033Search in Google Scholar

Yang, J. and Zhang, Y. (2015). Protein structure and function prediction using I-TASSER. Curr. Protoc. Bioinformatics 52, 5.8.1–5.815.10.1002/0471250953.bi0508s52Search in Google Scholar

Supplemental Material:

The online version of this article offers supplementary material (

Received: 2017-5-3
Accepted: 2017-8-31
Published Online: 2017-9-8
Published in Print: 2017-12-20

©2018 Walter de Gruyter GmbH, Berlin/Boston