Biochemical and biophysical studies to characterise the Ras:Sos:nucleotide interactions

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Abstract

Ras proteins are mutated in 30% of all human tumours contributing to several malignant phenotypes including abnormal cell growth, proliferation and apoptosis. The activity of Ras is controlled by the inter-conversion between GTP- and GDP- bound forms. This conversion is partly regulated by the binding of protein Son of Sevenless (Sos), a guanine nucleotide exchange factor. The mechanism of Ras activation via its interactions with Sos remains unclear making it challenging as an effective drug target. The aim of this work is to use Nuclear Magnetic Resonance (NMR) spectroscopy and other biophysical methods to understand the molecular activation of Ras via its interactions with Sos. In this thesis, the backbone and Cβ, as well as the partial side-chain NMR assignment for human K-Ras•GDP were completed at pH 7.4. We also revealed significant chemical shift differences between apo, GDP and GTPϒS-bound H-Ras states from the TROSY spectra. In addition, the monitoring of shift perturbations for H-Ras reveals several residues that appear to be central in Sos binding and may provide a starting point in the search for possible inhibition sites for future drug design. To gain a further understanding into the binding events of the Ras:Sos complex, we have expressed and purified the Sos construct containing the REM and Cdc25 domains (SosCat) for titration studies. Here, we have implemented a relatively novel approach to study large complexes (Stoffregen et al. 2012), by selectively labelling the [13C-] Met and Ile methyl groups of SosCat. This approach has provided an assignment for eight reporter signals. In addition, monitoring the shift perturbations of Met [13C-] methyls in the NMR spectra allowed us to examine individual residues at the two Ras binding sites (allosteric and catalytic sites) of SosCat. Disruption of H-Ras•GTPγS binding at the allosteric site (via SosCat W729E mutant) significantly weakens the interactions of Ras at the catalytic site. The data suggests a positive co-operative binding mechanism between the allosteric and catalytic sites, which is consistent with the allosteric feedback model. We have also measured the binding affinities of SosCat (by NMR spectroscopy and fluorescence) with wild type and Ras mutants using different GTP analogues. Our 15N-relaxation data of the H-Ras•GTPϒS:SosCat complex reveal dynamical changes in several regions of Ras other than the P-loop, switch I and II regions. In addition, the backbone NMR relaxation studies revealed that a complex between H-Ras•GTPϒS and SosCat proteins is dynamic and transiently formed. The reported work could be a significant step towards understanding the activation of Ras via its interactions with Sos; and in time the data may influence new anti-cancer treatments.

Layman's Abstract

The Ras family of small GTPases, which includes three isoforms; H-Ras, K-Ras and N-Ras, forms one of the most important nodal points in pathways targeted intensively in cancer drug discovery. These proteins are ubiquitously expressed in mammalian cells and regulate cell signalling pathways that are key regulators in cell growth, proliferation and differentiation. The activity of Ras is dependent upon the cycling between active GTP-bound state and inactive GDP-bound state. The activity of Ras is tightly controlled by its binding protein partners: GTPase activating proteins and guanine nucleotide exchange factors. One of the important research questions is the understanding of how Ras activity is regulated through its interactions with guanine nucleotide exchanges factors.Nuclear Magnetic resonance spectroscopy (NMR) is a biophysical technique that is used to detect structural changes of individual residues at the binding site interface of protein-protein interactions. The NMR spectrum of a protein gives rise to NMR signals that are then assigned to a specific residue of the protein. We have observed and assigned NMR signals from all the functionally important regions of Ras under physiological conditions. This has allowed us to identify binding site hotspots in the NMR spectra of Ras upon interactions with Sos. To gain a further understanding into the binding events of the Ras: Sos complex, we carried out a series of NMR-titration experiments, whereby increasing concentrations of Ras were added to Sos and analysed by NMR. Analysis of these NMR spectra enables us to monitor signals at the Ras: Sos binding sites under physiological conditions. Other objectives of this project are the use of further biophysical techniques such as fluorescent and Isothermal titration calorimetry to obtain binding affinities of Ras. In addition, we have also study the dynamics of the Ras:Sos complex and reveal regions of Ras that undergo different internal motions when in complex with Sos. This work could be a significant step towards understanding the molecular basis for activation of Ras via its interactions with cognate guanine nucleotide exchanges factors and, in time, the data may influence new anti-cancer treatments.

Keyword(s)

Ras; Sos

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