Improved Binding of Raf to Ras·GDP Is Correlated with Biological Activity

Christina Kiel,Daniel Filchtinski,Michael Spoerner,Gideon Schreiber,Hans Robert Kalbitzer,Christian Herrmann

doi:10.1074/jbc.m109.031153

Abstract

The GTP-binding protein Ras plays a central role in the regulation of various cellular processes, acting as a molecular switch that triggers signaling cascades. Only Ras bound to GTP is able to interact strongly with effector proteins like Raf kinase, phosphatidylinositol 3-kinase, and RalGDS, whereas in the GDP-bound state, the stability of the complex is strongly decreased, and signaling is interrupted. To determine whether this process is only controlled by the stability of the complex, we used computer-aided protein design to improve the interaction between Ras and effector. We challenged the Ras.Raf complex in this study because Raf among all effectors shows the highest Ras affinity and the fastest association kinetics. The proposed mutations were characterized as to their changes in dynamics and binding strength. We demonstrate that Ras-Raf interaction can only be improved at the cost of a loss in specificity of Ras.GTP versus Ras.GDP. As shown by NMR spectroscopy, the Raf mutation A85K leads to a shift of Ras switch I in the GTP-bound as well as in the GDP-bound state, thereby increasing the complex stability. In a luciferase-based reporter gene assay, Raf A85K is associated with higher signaling activity, which appears to be a mere matter of Ras-Raf affinity.

Highlights

IntroductionRas has a large number of effector proteins, such as Raf kinase [5, 6], RalGDS [7], phosphatidylinositol 3-kinase (8 –10), and Nore1A [11, 12], representing different signal directions
We have demonstrated that PARE-predicted mutations around the Ras binding domain (RBD) of the effector RalGDS transformed it into a Raf-like effector, efficiently binding Ras [32]
Lysine residues were introduced at these positions into the crystal structure of the Raps/Raf-RBD complex (Protein Data Bank entry 1GUA; Ras homologue Raps loaded with the non-hydrolyzable GTP analogue GppNHp and complexed with the Raf-RBD wild type) using SwissPdbViewer [37]

Summary

Introduction

Ras has a large number of effector proteins, such as Raf kinase [5, 6], RalGDS [7], phosphatidylinositol 3-kinase (8 –10), and Nore1A [11, 12], representing different signal directions These effectors have in common the so-called Ras binding domain (RBD), enabling them to interact with Ras. Only the GTP-bound form of Ras binds strongly to effectors and leads to their activation. Except Nore1A with a small dissociation rate constant, koff [16], the values for this constant are similar for the other effectors (ϳ10 sϪ1), with the differences in affinity being governed by variations in kon (17, 20 –22) Another common feature observed in all Ras1⁄7RBD complex structures is the high charge complementarity between the two proteins. Our results allow intriguing insight into the molecular basis of Ras/effector binding specificity and activation

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Results

Conclusion