Recognizing and defining true Ras binding domains I: biochemical analysis.
Common domain databases contain sequence motifs which belong to the ubiquitin fold family and are called Ras binding (RB) and Ras association (RalGDS/AF6 Ras associating) (RA) domains. The name implies that they bind to Ras (or Ras-like) GTP-binding proteins, and a few of them have been documented to qualify as true Ras effectors, defined as binding only to the activated GTP-bound form of Ras. Here we have expressed a large number of these domains and investigated their interaction with Ras, Rap and M-Ras. While their (albeit weak) sequence homology suggest that the domains adopt a common fold, not all of them bind to Ras proteins, irrespective of whether they are called RB or RA domains. We used fluorescence spectroscopy and isothermal titration calorimetry to show that the binding affinities vary over a large range, and are usually specific for either Ras or Rap. Moreover, the specificity is dictated by a set of key residues in the interface. Stopped-flow kinetic analysis showed that the association rate constants determine the different affinities of effector binding, while the dissociation rate constants are in a similar range. Manual sequence analysis allowed us to define positively charged sequence epitopes in certain secondary structure elements of the ubiquitin fold (beta1, beta2 and alpha1) which are located at similar positions and comprise the hot spots of the binding interface. These residues are important to qualify an RA/RB domain as a true candidate Ras or Rap effector.
Wohlgemuth
Sabine
S
Max-Planck-Institut für Molekulare Physiologie, Abteilung Strukturelle Biologie, Otto-Hahn Str. 11, Dortmund 44227, Germany.
Kiel
Christina
C
Krämer
Astrid
A
Serrano
Luis
L
Wittinghofer
Fred
F
Herrmann
Christian
C
D000818:Animals; D002151:Calorimetry; D002942:Circular Dichroism; D004259:DNA-Directed DNA Polymerase; D000939:Epitopes; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D020827:rap1 GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D018631:ras Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000939:Epitopes; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase; D020827:rap1 GTP-Binding Proteins; D018631:ras Proteins