The RAS-Effector Interface: Isoform-Specific Differences in the Effector Binding Regions

PLoS One. 2016 Dec 9;11(12):e0167145. doi: 10.1371/journal.pone.0167145. eCollection 2016.

Abstract

RAS effectors specifically interact with the GTP-bound form of RAS in response to extracellular signals and link them to downstream signaling pathways. The molecular nature of effector interaction by RAS is well-studied but yet still incompletely understood in a comprehensive and systematic way. Here, structure-function relationships in the interaction between different RAS proteins and various effectors were investigated in detail by combining our in vitro data with in silico data. Equilibrium dissociation constants were determined for the binding of HRAS, KRAS, NRAS, RRAS1 and RRAS2 to both the RAS binding (RB) domain of CRAF and PI3Kα, and the RAS association (RA) domain of RASSF5, RALGDS and PLCε, respectively, using fluorescence polarization. An interaction matrix, constructed on the basis of available crystal structures, allowed identification of hotspots as critical determinants for RAS-effector interaction. New insights provided by this study are the dissection of the identified hotspots in five distinct regions (R1 to R5) in spite of high sequence variability not only between, but also within, RB/RA domain-containing effectors proteins. Finally, we propose that intermolecular β-sheet interaction in R1 is a central recognition region while R3 may determine specific contacts of RAS versus RRAS isoforms with effectors.

MeSH terms

  • Amino Acid Sequence
  • Binding Sites / genetics
  • Binding, Competitive
  • Carrier Proteins / chemistry
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Class I Phosphatidylinositol 3-Kinases
  • GTP Phosphohydrolases / chemistry
  • GTP Phosphohydrolases / genetics
  • GTP Phosphohydrolases / metabolism*
  • Humans
  • Kinetics
  • Membrane Proteins / chemistry
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Models, Molecular
  • Monomeric GTP-Binding Proteins / chemistry
  • Monomeric GTP-Binding Proteins / genetics
  • Monomeric GTP-Binding Proteins / metabolism*
  • Phosphatidylinositol 3-Kinases / chemistry
  • Phosphatidylinositol 3-Kinases / genetics
  • Phosphatidylinositol 3-Kinases / metabolism
  • Protein Binding
  • Protein Domains
  • Protein Structure, Secondary
  • Proto-Oncogene Proteins p21(ras) / chemistry
  • Proto-Oncogene Proteins p21(ras) / genetics
  • Proto-Oncogene Proteins p21(ras) / metabolism*
  • Sequence Homology, Amino Acid
  • Signal Transduction
  • ral Guanine Nucleotide Exchange Factor / chemistry
  • ral Guanine Nucleotide Exchange Factor / genetics
  • ral Guanine Nucleotide Exchange Factor / metabolism

Substances

  • Carrier Proteins
  • KRAS protein, human
  • Membrane Proteins
  • RASSF5 protein, human
  • ral Guanine Nucleotide Exchange Factor
  • Phosphatidylinositol 3-Kinases
  • Class I Phosphatidylinositol 3-Kinases
  • PIK3CA protein, human
  • RRAS2 protein, human
  • GTP Phosphohydrolases
  • NRAS protein, human
  • HRAS protein, human
  • Monomeric GTP-Binding Proteins
  • Proto-Oncogene Proteins p21(ras)

Grant support

This study was supported in part by the German Research Foundation (Deutsche Forschungsgemeinschaft or DFG) through the Collaborative Research Center 974 (SFB 974) “Communication and Systems Relevance during Liver Injury and Regeneration”, the International Research Training Group 1902 (IRTG 1902) “Intra- and interorgan communication of the cardiovascular system”, the German Federal Ministry of Education and Research (BMBF) - German Network of RASopathy Research (GeNeRARe; fund number 01GM1519D), and the European Network on Noonan Syndrome and Related Disorders (NSEuroNet; fund number 01GM1602B).