Modulation of the Switch Regions in Ras GTPase

Ras is a monomeric GTPase which in its GTP‐bound form can bind various effectors in signal transduction pathways. Through its interaction with the effector Raf and associated Ras/Raf/MEK/ERK cascade it is directly involved in the control of cell proliferation and thus implicated in many human cancers. Switch I and switch II undergo conformational transition during hydrolysis and contains active site residues and the effector binding sites. In the Ras/Raf complex only switch I is involved in the interaction. We have a crystal form of Ras in which switch I has the conformation seen in its complex with Raf and switch II is disordered and free of crystal contacts. This facilitated discovery of an allosteric switch, activated in our crystals by calcium acetate, that places the catalytic residue Q61 in the active site. In this crystal form Ras can adopt both the active and inactive states of the allosteric switch, where the activated state is characterized by a shift in helix3/loop7 toward helix 4 and ordering of switch II for catalytic activity. Here we show that DTT stabilizes the inactive form of the switch by occupying a pocket between switch II and helix 3, resulting in an ordered anti‐catalytic conformation of switch II. This anti‐catalytic conformation is also seen in highly transforming mutants, such as RasQ61L. By changing the relative concentrations of calcium acetate and DTT in Ras crystal soaks we show transition between the on and off states of the allosteric switch. Our results provide a proof of concept that the allosteric site and switch II/helix3 pocket can be used to modulate the state of Ras and should therefore serve as novel targets sites in ligand design. NIH R01 CA096867