Ras-Efektör Etkileşimlerinin Yapısal Detaylarının Açığa Çıkarılması

Yıl 2019, Cilt: 31 Sayı: 1, 90 - 99, 31.03.2019

Serena Muratcıoğlu Saliha Ece Acuner Özbabacan

https://doi.org/10.7240/jeps.528662

Öz

Hücre zarıyla ilintili küçük Ras proteinleri pek
çok efektöre bağlanıp onları aktif hale getirerek hücre çoğalması, göçü,
hayatta kalma ve farklılaşması gibi çeşitli hücresel işlevleri kontrol ederler.
Ras üzerindeki mutasyonlar, yapısal olarak aktif proteine sebebiyet verir ve
insandaki birçok kanser tipinde tespit edilmişlerdir ve Ras topluluğu Ras’ı
hedef alan küçük moleküllü inhibitörler tasarlamak yerine Ras’ın efektör
yolaklarındaki protein-protein etkileşimlerini hedef alarak Ras’ın zar üzerindeki
lokalizasyonunu engellemeyi amaçlamaktadır. Ras’ın en çok çalışılan
efektörleri, Raf, PI3K ve RalGDS, Ras’a aynı yüzeyden bağlanmasına rağmen karşıt
sinyal yolaklarını ortaya çıkarırlar ve dolayısıyla hücrenin bu yolaklar
arasındaki zamansal ve mekansal kararları kritik öneme sahiptir. Ras/efektör
etkileşimlerinin yapısal detaylarını açığa çıkarmak, hücrenin karar
mekanizmasını anlamamıza ve protein-protein etkileşimlerini hassas olarak
hedeflememize yardımcı olabilir. Bununla birlikte, sadece birkaç Ras/efektör
kompleksinin kristal yapısı PDB'de bulunmaktadır. Bu çalışmada, Ras/efektör
komplekslerinin 3 boyutlu yapıları PRISM algoritması ile modellenmiştir ve Ras
üzerindeki sıcak nokta kalıntılarının yanı sıra önemli bağlanma bölgeleri
belirlenmiştir. Efektörler ayrıca, rekabetçi yolları ve "efektör
lobu" dışındaki bağlayıcı bölgeleri belirlemek için Ras'daki bağlayıcı
bölgelere göre sınıflandırılmıştır. Modellenen kompleksler, Ras ve ortakları
arasındaki arayüzeyler hakkında onkojenik Ras sinyal iletimini bloke etmek için
ilaç tasarım çalışmalarına rehberlik etme potansiyeli olan önemli bilgiler
ortaya koymaktadır.

Anahtar Kelimeler

Ras efektörleri, onkojenik sinyal iletimi, protein-protein etkileşimi, protein-protein arayüzeyi, protein yapısı

Elucidating Structural Details of Ras-Effector Interactions

Öz

Small
membrane-associated Ras proteins mediate a wide range of cellular functions, such as cell proliferation, migration,
survival, and differentiation; through binding
and activating numerous effectors. Constitutively
active mutant Ras proteins are detected in various types of human cancer and Ras community seeks approaches other
than small-molecule Ras inhibitors; such as targeting the protein-protein
interactions in the downstream Ras effector pathways and preventing its membrane localization. Although
the most studied effectors of Ras, i.e. Raf, PI3K and RalGDS, bind Ras through the
same site, they elicit opposing signaling pathways and thus, the temporal and
spatial decision of the cell among them is critical. Elucidating the structural
details of Ras/effector interactions can help us understand the cell decision
and target the protein-protein interactions precisely. However, only a few crystal
structures of Ras in complex with an effector are deposited in PDB. Here, the
3D structures of Ras/effector complexes were modeled with the PRISM algorithm and important binding sites as well as hot spot
residues on Ras were identified. The effectors were also classified according
to the binding regions on Ras, to determine the competitive pathways and the
binding regions other than the “effector lobe”. The
modeled complexes reveal important information about the interfaces between Ras
and its partners with the potential of guiding drug design studies to block oncogenic Ras signaling.

Anahtar Kelimeler

Ras effectors, oncogenic signaling, protein-protein interaction, protein-protein interface, hot spot, protein structure

Kaynakça

• [1] Campbell, S. L., Khosravi-Far, R., Rossman, K. L., Clark, G. J., & Der, C. J. (1998). Increasing complexity of Ras signaling. Oncogene, 17(11 Reviews), 1395-1413.
• [2] Reuther, G. W., & Der, C. J. (2000). The Ras branch of small GTPases: Ras family members don't fall far from the tree. Curr Opin Cell Biol, 12(2), 157-165.
• [3] Cherfils, J., & Zeghouf, M. (2013). Regulation of small GTPases by GEFs, GAPs, and GDIs. Physiol Rev, 93(1), 269-309.
• [4] Geyer, M., & Wittinghofer, A. (1997). GEFs, GAPs, GDIs and effectors: taking a closer (3D) look at the regulation of Ras-related GTP-binding proteins. Curr Opin Struct Biol, 7(6), 786-792.
• [5] Sprang, S. R. (1997). G proteins, effectors and GAPs: structure and mechanism. Curr Opin Struct Biol, 7(6), 849-856.
• [6] Reuther, G. W., & Der, C. J. (2000). The Ras branch of small GTPases: Ras family members don’t fall far from the tree. Current opinion in cell biology, 12(2), 157-165.
• [7] Takai, Y., Sasaki, T., & Matozaki, T. (2001). Small GTP-binding proteins. Physiological reviews, 81(1), 153-208.
• [8] Muegge, I., Schweins, T., Langen, R., & Warshel, A. (1996). Electrostatic control of GTP and GDP binding in the oncoprotein p21ras. Structure, 4(4), 475-489.
• [9] Muratcioglu, S., Chavan, T. S., Freed, B. C., et al. (2015). GTP-Dependent K-Ras Dimerization. Structure, 23(7), 1325-1335.
• [10] Lu, S., Jang, H., Muratcioglu, S., et al. (2016). Ras Conformational Ensembles, Allostery, and Signaling. Chem Rev, 116(11), 6607-6665.
• [11] Gorfe, A. A., Grant, B. J., & McCammon, J. A. (2008). Mapping the nucleotide and isoform-dependent structural and dynamical features of Ras proteins. Structure, 16(6), 885-896.
• [12] Buhrman, G., O'Connor, C., Zerbe, B., et al. (2011). Analysis of binding site hot spots on the surface of Ras GTPase. J Mol Biol, 413(4), 773-789.
• [13] Buhrman, G., Holzapfel, G., Fetics, S., & Mattos, C. (2010). Allosteric modulation of Ras positions Q61 for a direct role in catalysis. Proc Natl Acad Sci U S A, 107(11), 4931-4936.
• [14] Barbacid, M. (1987). ras genes. Annu Rev Biochem, 56, 779-827.
• [15] Bos, J. L. (1989). ras oncogenes in human cancer: a review. Cancer Res, 49(17), 4682-4689.
• [16] Rojas, A. M., Fuentes, G., Rausell, A., & Valencia, A. (2012). The Ras protein superfamily: evolutionary tree and role of conserved amino acids. J Cell Biol, 196(2), 189-201.
• [17] Forbes, S. A., Bindal, N., Bamford, S., et al. (2011). COSMIC: mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer. Nucleic Acids Res, 39(Database issue), D945-950.
• [18] Karnoub, A. E., & Weinberg, R. A. (2008). Ras oncogenes: split personalities. Nat Rev Mol Cell Biol, 9(7), 517-531.
• [19] Cox, A. D., Fesik, S. W., Kimmelman, A. C., Luo, J., & Der, C. J. (2014). Drugging the undruggable RAS: Mission possible? Nat Rev Drug Discov, 13(11), 828-851.
• [20] Maurer, T., Garrenton, L. S., Oh, A., et al. (2012). Small-molecule ligands bind to a distinct pocket in Ras and inhibit SOS-mediated nucleotide exchange activity. Proc Natl Acad Sci U S A, 109(14), 5299-5304.
• [21] Ostrem, J. M., Peters, U., Sos, M. L., Wells, J. A., & Shokat, K. M. (2013). K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions. Nature, 503(7477), 548-551.
• [22] Shima, F., Yoshikawa, Y., Ye, M., et al. (2013). In silico discovery of small-molecule Ras inhibitors that display antitumor activity by blocking the Ras-effector interaction. Proc Natl Acad Sci U S A, 110(20), 8182-8187.
• [23] Sun, Q., Burke, J. P., Phan, J., et al. (2012). Discovery of small molecules that bind to K-Ras and inhibit Sos-mediated activation. Angew Chem Int Ed Engl, 51(25), 6140-6143.
• [24] Athuluri-Divakar, S. K., Vasquez-Del Carpio, R., Dutta, K., et al. (2016). A Small Molecule RAS-Mimetic Disrupts RAS Association with Effector Proteins to Block Signaling. Cell, 165(3), 643-655.
• [25] Cruz-Migoni, A., Canning, P., Quevedo, C. E., et al. (2019). Structure-based development of new RAS-effector inhibitors from a combination of active and inactive RAS-binding compounds. Proc Natl Acad Sci U S A.
• [26] Engin, H. B., Carlin, D., Pratt, D., & Carter, H. (2017). Modeling of RAS complexes supports roles in cancer for less studied partners. BMC Biophys, 10(Suppl 1), 5.
• [27] Wittinghofer, A., & Herrmann, C. (1995). Ras-effector interactions, the problem of specificity. FEBS Lett, 369(1), 52-56.
• [28] Marshall, C. J. (1996). Ras effectors. Curr Opin Cell Biol, 8(2), 197-204.
• [29] McCormick, F., & Wittinghofer, A. (1996). Interactions between Ras proteins and their effectors. Curr Opin Biotechnol, 7(4), 449-456.
• [30] Koide, H., Satoh, T., Nakafuku, M., & Kaziro, Y. (1993). GTP-dependent association of Raf-1 with Ha-Ras: identification of Raf as a target downstream of Ras in mammalian cells. Proc Natl Acad Sci U S A, 90(18), 8683-8686.
• [31] Spoerner, M., Herrmann, C., Vetter, I. R., Kalbitzer, H. R., & Wittinghofer, A. (2001). Dynamic properties of the Ras switch I region and its importance for binding to effectors. Proceedings of the National Academy of Sciences, 98(9), 4944-4949.
• [32] Mott, H. R., & Owen, D. (2015). Structures of Ras superfamily effector complexes: What have we learnt in two decades? Crit Rev Biochem Mol Biol, 50(2), 85-133.
• [33] Kolch, W. (2000). Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions. Biochem J, 351 Pt 2, 289-305.
• [34] Pacold, M. E., Suire, S., Perisic, O., et al. (2000). Crystal structure and functional analysis of Ras binding to its effector phosphoinositide 3-kinase γ. Cell, 103(6), 931-944.
• [35] Rodriguez-Viciana, P., & McCormick, F. (2005). RalGDS comes of age. Cancer Cell, 7(3), 205-206.
• [36] Datta, S. R., Brunet, A., & Greenberg, M. E. (1999). Cellular survival: a play in three Akts. Genes & development, 13(22), 2905-2927.
• [37] Khwaja, A., Rodriguez‐Viciana, P., Wennström, S., Warne, P. H., & Downward, J. (1997). Matrix adhesion and Ras transformation both activate a phosphoinositide 3‐OH kinase and protein kinase B/Akt cellular survival pathway. The EMBO journal, 16(10), 2783-2793.
• [38] Feig, L. A., Urano, T., & Cantor, S. (1996). Evidence for a Ras/Ral signaling cascade. Trends in biochemical sciences, 21(11), 438-441.
• [39] Hofer, F., Fields, S., Schneider, C., & Martin, G. S. (1994). Activated Ras interacts with the Ral guanine nucleotide dissociation stimulator. Proceedings of the National Academy of Sciences, 91(23), 11089-11093.
• [40] Marais, R., Light, Y., Paterson, H., & Marshall, C. (1995). Ras recruits Raf‐1 to the plasma membrane for activation by tyrosine phosphorylation. The EMBO journal, 14(13), 3136-3145.
• [41] Yordy, J. S., & Muise-Helmericks, R. C. (2000). Signal transduction and the Ets family of transcription factors. Oncogene, 19(55), 6503.
• [42] Vojtek, A. B., Hollenberg, S. M., & Cooper, J. A. (1993). Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell, 74(1), 205-214.
• [43] Chuang, E., Barnard, D., Hettich, L., Zhang, X. F., Avruch, J., & Marshall, M. S. (1994). Critical binding and regulatory interactions between Ras and Raf occur through a small, stable N-terminal domain of Raf and specific Ras effector residues. Mol Cell Biol, 14(8), 5318-5325.
• [44] Ghosh, S., & Bell, R. M. (1994). Identification of discrete segments of human Raf-1 kinase critical for high affinity binding to Ha-Ras. J Biol Chem, 269(49), 30785-30788.
• [45] Gohlke, H., Kiel, C., & Case, D. A. (2003). Insights into protein-protein binding by binding free energy calculation and free energy decomposition for the Ras-Raf and Ras-RalGDS complexes. J Mol Biol, 330(4), 891-913.
• [46] Krygowska, A. A., & Castellano, E. (2018). PI3K: a crucial piece in the RAS signaling puzzle. Cold Spring Harbor perspectives in medicine, 8(6), a031450.
• [47] Fetics, S. K., Guterres, H., Kearney, B. M., et al. (2015). Allosteric effects of the oncogenic RasQ61L mutant on Raf-RBD. Structure, 23(3), 505-516.
• [48] Aytuna, A. S., Gursoy, A., & Keskin, O. (2005). Prediction of protein-protein interactions by combining structure and sequence conservation in protein interfaces. Bioinformatics, 21(12), 2850-2855.
• [49] Ogmen, U., Keskin, O., Aytuna, A. S., Nussinov, R., & Gursoy, A. (2005). PRISM: protein interactions by structural matching. Nucleic Acids Res, 33(Web Server issue), W331-336.
• [50] Tuncbag, N., Gursoy, A., Nussinov, R., & Keskin, O. (2011). Predicting protein-protein interactions on a proteome scale by matching evolutionary and structural similarities at interfaces using PRISM. Nat Protoc, 6(9), 1341-1354.
• [51] Tuncbag, N., Keskin, O., Nussinov, R., & Gursoy, A. (2012). Fast and accurate modeling of protein-protein interactions by combining template-interface-based docking with flexible refinement. Proteins, 80(4), 1239-1249.
• [52] Mashiach, E., Nussinov, R., & Wolfson, H. J. (2010). FiberDock: Flexible induced-fit backbone refinement in molecular docking. Proteins, 78(6), 1503-1519.
• [53] Zhang, Y. (2008). I-TASSER server for protein 3D structure prediction. BMC Bioinformatics, 9, 40.
• [54] Cukuroglu, E., Gursoy, A., & Keskin, O. (2012). HotRegion: a database of predicted hot spot clusters. Nucleic Acids Res, 40(Database issue), D829-833.
• [55] Feig, L. A., & Buchsbaum, R. J. (2002). Cell signaling: life or death decisions of ras proteins. Curr Biol, 12(7), R259-261.
• [56] Mitin, N., Konieczny, S. F., & Taparowsky, E. J. (2006). RAS and the RAIN/RasIP1 effector. Methods Enzymol, 407, 322-335.
• [57] Choy, E., Chiu, V. K., Silletti, J., et al. (1999). Endomembrane trafficking of ras: the CAAX motif targets proteins to the ER and Golgi. Cell, 98(1), 69-80.
• [58] Liedtke, M., Ayton, P. M., Somervaille, T. C., Smith, K. S., & Cleary, M. L. (2010). Self-association mediated by the Ras association 1 domain of AF6 activates the oncogenic potential of MLL-AF6. Blood, 116(1), 63-70.
• [59] Smith, M. J., Ottoni, E., Ishiyama, N., et al. (2017). Evolution of AF6-RAS association and its implications in mixed-lineage leukemia. Nat Commun, 8(1), 1099.