PhD Scientific Days 2018

Budapest, April 19–20, 2018

Understanding the role of dynamics in function of oncogenic KRas mutants

Pálfy, Gyula

Gyula Pálfy, István Vida and András Perczel
Laboratory of Structural Chemistry and Biology, Eötvös L. University, Budapest

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Text of the abstract

Oncogenic variants of KRAS gene are among the most important mutations causing cancer in human.1 KRas protein, encoded by this gene, is a membrane-bound small GTPase which acts as a molecular switch and plays a key role in many signal transduction pathways regulating cell proliferation, differentiation and survival. It alternates between two forms: the GTP-bound active and the GDP-bound inactive conformers. Activation and inactivation of KRas is controlled by the guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), respectively.2 The most frequent mutant KRas-G12C is ”frozen” in its active state. Switch-I and -II regions, which have crucial part in the autohydrolysis mechanism of KRas, are known to have dynamic behavior. Although, the backbone dynamics of HRas is well-described at multiple time scales with different NMR techniques, KRas and its mutants have hardly been investigated.3 Here we are providing backbone dynamics (ps-ns time scale) of both KRas and its oncogenic mutant KRas-G12C by using 1H-NMR measurements (T1, T2, HetNOE) at physiological pH and 298 K in its GDP- and GTP-bound forms.
Catalytic domain of KRas and KRas-G12C were expressed and purified as follows: pET-15b vectors coding the proteins were transformed into E. coli BL21 (DE3) competent cells and their 15N-labeled form were expressed. After sonication and centrifugation, proteins were purified by Ni2+-ion metal affinity and size-exclusion chromatography.
Measurements were completed on a Bruker Avance III 700 MHz spectrometer using prodigy probe head. Data were analyzed using the reduced spectral density mapping method as well as Lipari-Szabó model-free analysis. We found that Switch-I and -II regions are highly dynamic showing Rex exchange in Switch-I region, while mutation in the P-loop alters its mobility. We found that in KRas-G12C this region is more mobile compared to the wild type form. Comparing the dynamics of GTP- and GDP-bound forms of KRas-G12C, we found broadened resonances in the P-loop, Switch-I and -II regions due to the increased NH mobility of selected residues also detected in HRas.4 However, the behavior of Y32 of Switch-I is opposite, namely according to HetNOE measurements it is more rigid in its GTP-bound form, described here for the first time. This reveals the important role of Y32 in hydrolyzing GTP. According to our results protein dynamics investigated by NMR spectroscopy can play a key role in understanding GTP hydrolysis mechanism as well as mutant specific properties of KRas oncogene mutants. This work is supported by NVKP-16-1-2016-0020.

1. Hunter, J. C. et al. Biochemical and structural analysis of common cancer-associated KRAS mutations Mol. Canc. Res. 13 (9), 1325-1335 (2015).
2. Ostrem, J. M. L. & Shokat, K. M. Direct small-molecule inhibitors of KRAS: from structural insights to mechanism-based design Nat. Rew. 15, 771-785 (2016).
3. Buhrman, G. et al. Analysis of binding site hot spots on the surface of Ras GTPase J. Mol. Biol. 413(4), 773-789 (2011).
4. Ito, Y. et al. Regional polysterism in the GTP-bound form of the human c-Ha-Ras protein Biochemistry 36(30) 9109-9119 (1997).