GTP-Ras disrupts the intramolecular complex of C1 and RA domains of Nore1.
The novel Ras effector mNore1, capable of inducing apoptosis, is a multidomain protein. It comprises a C1 domain homologous to PKC and an RA domain similar to the Ras effectors AF-6 and RalGDS. Here, we determine the affinity of these two domains to the active forms of Ras and Rap1 using isothermal calorimetric titration. The interaction of Ras/Rap1-GTP with the RA domain of mNore1 is weakened significantly by direct binding of the C1 domain to the RA domain. In order to analyze this observation in atomic detail, we solved the C1 solution structure by NMR. By determining chemical shifts and relaxation rates, we can show an intramolecular complex of C1-RA. GTP-Ras titration and binding to RA disrupts this complex and displaces the C1 domain. Once the C1 domain tumbles freely in solution, a lipid binding interface becomes accessible. Furthermore, we provide evidence of phosphatidylinositol 3-phosphate binding of the free C1 domain.
Harjes
Elena
E
Molecular and Structural Biophysics, Max-Planck Institute of Molecular Physiology, Otto-Hahn Strasse 11, D-44227 Dortmund, Germany.
Harjes
Stefan
S
Wohlgemuth
Sabine
S
Müller
Karl-Heinz
KH
Krieger
Elmar
E
Herrmann
Christian
C
Bayer
Peter
P
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D008055:Lipids / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D020827:rap1 GTP-Binding Proteins / Q000737:chemistry*; D018631:ras Proteins / Q000737:chemistry*
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D008055:Lipids; D018129:Phosphatidylinositol Phosphates; C455831:Rassf5 protein, mouse; C055525:phosphatidylinositol 3-phosphate; D020827:rap1 GTP-Binding Proteins; D018631:ras Proteins