Using a combination of methods, a team of scientists from Ruhr University has reported that the binding partner of Ras in its bonded form does not contain any hydrogen atoms in the phosphate groups. Ras plays a role in a variety of cell signaling pathways including cell proliferation, and a better understanding of the protonation states of GTP and GppNHp in Ras proteins could help advance cancer research. Details on the methods and findings were published last week in the Journal of Biological Chemistry.
Ras proteins function as small switches: depending on whether they are bonded with guanosine triphosphate (GTP) or with guanosine diphosphate (GDP), they are either switched on or off. The GTP bond triggers complex signaling pathways that lead to the nucleus where they initiate cell division.
If this process is inhibited, for example due to mutations in the Ras protein, Ras remains in its active state, and the ongoing division of cells can lead to the formation of a tumor. "More than 30 per cent of all tumors carry a mutation of the Ras protein," explained Daniel Mann, first author of the paper. "Consequently, the cleavage reaction of GTP in Ras is the key for understanding many types of cancer."
In order to understand GTP cleavage reactions in Ras, a precise image of the starting point is needed, which is difficult if not impossible to obtain with conventional methods such as X-ray crystallography. It has previously been assumed that the phosphate groups of GTP-bound Ras were free of hydrogen atoms.
Using neutron diffraction analyses of the GTP-analogue GppNHp, the Ruhr University team recorded both the infrared spectrums of the GppNHp that is deployed in neutron diffraction and the spectrums of the natural GTP in Ras. As a result, it was possible to draw a direct comparison with neutron diffraction, as well as a comparison with an environment that resembles that of the human cell. "The measured infrared data enable us to decode molecular reactions at the highest temporal and spatial resolution," noted Klaus Gerwert, a corresponding author on the paper. "However, the information is coded in infrared spectra and is therefore difficult to interpret."
The team thus used a process for decoding that facilitated the calculation of experimental data such as infrared spectra and nuclear spin spectra gathered through coupled quantum mechanics/molecular mechanics simulations. "If the calculated spectra correspond with the spectra measured in experiments, we can translate the figures we measured in experiments into three-dimensional films," Mann explained.
"These precise results have enabled us to finally determine the accurate protonation state of GTP bound to Ras proteins: the GTP phosphate groups do indeed not carry any hydrogen atoms," concluded Gerwert.