Structural Studies of Membrane-tethered KRAS4b Interaction with RAF and Inhibitors, and RIT1 Disease Mutants

• Main Author: Fang, Zhenhao
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2020
• Subjects: Biochemistry; Biophysics; Molecular biology
• ISBN: 9798662393523

For more than three decades, RAS genes have been recognized as among the most important cancer-causing genes. RAS mutations are found in some of the most aggressive and deadly cancers, and are generally associated with poor prognosis. However decades of research have yet to yield a drug that can effectively inhibit RAS. Failed attempts to develop RAS inhibitors, including GTP-competitive analogs and farnesyltransferase inhibitors, have driven researchers from this field and led pharmaceutical companies to abandon RAS projects. However, in the last few years, researchers have started returning to this field, recognizing that RAS is still one of the most important targets in oncology. Although now armed with improved technology and a better understanding of RAS, researchers are mindful that there are still many hurdles, including the knowledge gap in understanding bilayer-anchored KRAS. RAF is one of the most important effectors of RAS and membrane anchoring of KRAS is critical for RAF activation. The RAS-binding domain (RBD) and Cysteine-rich domain (CRD) of RAF are key elements coupling RAS GTP loading and RAF activity. Their engagement with KRAS-GTP and membrane relieves the auto-inhibition of RAF kinase domain. This proposal aims to improve understanding of structure and function of KRAS in complex with CRAF RBD-CRD on biological membranes, and develop novel approaches towards inhibiting RAS in its membrane-tethered form by exploiting its tendancy to adopt an ‘occluded’ orientation on the bilayer surface. The discovery of RAS oncogene in human cancer marked the beginning of decades' journey of exploring the structure and function of RAS superfamily members. Recently, the RAS-like wildly expressed in many tissues (RIT1) came to researchers' spotlight because of its etiologic role in Noonan Syndrome and cancer. Using a real-time NMR-based GTPase assay, we characterized the GTP hydrolysis and nucleotide exchange of RIT1 WT and five disease-associated mutants including S35T, A57G, Y89H, F82V and T83P. This characterization will facilitate understanding of the biophysical properties and disease role of RIT1 and its mutants.