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 (Ras(on)GTP) and enables effector binding, which then activates signal-transduction pathways. GTP hydrolysis, catalysed by Ras and GAP, returns the signal to "off" (Ras(off)GDP). Oncogenic mutations in Ras prevent this hydrolysis, and thereby cause uncontrolled cell growth. In the Ras(off)-to-Ras(on) 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 Ras(off) to Ras(on) a GTP-bound intermediate is now identified, in which effector binding is still prevented (Ras(off)GTP). The loop movement from Ras(off)GTP to Ras(on)GTP 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 Ras(on)GTP state towards the Ras(off)GTP 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.
Kötting
Carsten
C
Lehrstuhl für Biophysik, Ruhr-Universität Bochum, 44780 Bochum, Germany. carsten.koetting@rub.de
Kallenbach
Angela
A
Suveyzdis
Yan
Y
Eichholz
Carolin
C
Gerwert
Klaus
K
D001608:Beryllium / Q000737:chemistry; D005459:Fluorides / Q000737:chemistry; D008958:Models, Molecular; D011485:Protein Binding; D017550:Spectroscopy, Fourier Transform Infrared; D013499:Surface Properties; D018631:ras Proteins / Q000737:chemistry / Q000378:metabolism*
C035982:beryllium fluoride; D018631:ras Proteins; D001608:Beryllium; D005459:Fluorides