Surface Change of Ras Enabling Effector Binding Monitored in Real Time at Atomic Resolution

Ras, the prototype of the Ras superfamily, acts as a molecular switch for cell growth. External growth signals induce a GDP‐to‐GTP exchange. This modifies the Ras surface (RasonGTP) and enables effector binding, which then activates signal‐transduction pathways. GTP hydrolysis, catalysed by Ras and GAP, returns the signal to “off” (RasoffGDP). Oncogenic mutations in Ras prevent this hydrolysis, and thereby cause uncontrolled cell growth. In the Rasoff‐to‐Rason transition, the Ras surface is changed by a movement of the switch I loop that controls effector binding. We monitored this surface change at atomic resolution in real time by time‐resolved FTIR (trFTIR) spectroscopy. In the transition from Rasoff to Rason a GTP‐bound intermediate is now identified, in which effector binding is still prevented (RasoffGTP). The loop movement from RasoffGTP to RasonGTP was directly monitored by the CO vibration of Thr35. The structural change creates a binding site with a rate constant of 5 s−1 at 260 K. A small molecule that shifted the equilibrium from the RasonGTP state towards the RasoffGTP state would prevent effector binding, even if hydrolysis were blocked by oncogenic mutations. We present a spectroscopic fingerprint of both states that can be used as an assay in drug screening for such small molecules. RasoffGTP. When GTP is bound to Ras the molecular switch is usually “on”; however, in the RasoffGTP state characterized here, it is “off” and signal transduction is blocked. This state and the time course of the switching process were investigated by FTIR spectroscopy at the atomic level by using para‐hydroxyphenacyl‐caged GTP and Ras labelled with a threonine isotope.