Chemical and Biological Engineering PhD Thesis Defense by Serena Muratcıoğlu

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KOÇ UNIVERSITY

GRADUATE SCHOOL OF SCIENCES & ENGINEERING

CHEMICAL AND BIOLOGICAL ENGINEERING

PhD THESIS DEFENSE BY SERENA MURATCIOĞLU

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Title: Computational and Experimental Investigation of Ras Homodimer Formation, Ras-effector Interactions and Ras Shuttling

Speaker: Serena Muratcıoğlu

Time: August 03, 2017, 13:00 pm

Place: ENG208

Koç University

Rumeli Feneri Yolu

Sariyer, Istanbul

Thesis Committee Members:

Prof. Özlem Keskin (Advisor, Koc University)

Prof. Attila Gürsoy (Co-advisor, Koc University)

Prof. İ. Halil Kavaklı (Koc University)

Assoc. Prof. Elif Özkırımlı Ölmez (Bogazici University)

Assoc. Prof. Gizem Dinler Doğanay (Istanbul Technical University)

Assist. Prof. Cem Albayrak (Koc University)

Abstract:

Ras proteins recruit and activate effectors, including Raf and PI3K, that transmit receptor-initiated signals. Activation of Ras elicits a wide variety of cellular responses depending on the specific effector that becomes activated. Ras signaling cascades are still not entirely understood. Among the pressing open questions are how the effectors are regulated, and how the cell decides among them under different environments and in distinct cancers. Ras is believed to function as a monomer. Monomeric Ras can bind Raf; however, activation of Raf requires its dimerization. Raf alone does not form a stable dimer. Thus, it was suspected that dimeric Ras may promote dimerization and activation of Raf. How dimerization relates to activation of effectors, however, is still unclear. In this dissertation, I combined experimental and computational approaches to improve the current understanding of the structural basis for Ras dimerization and to investigate the effects of dimerization on effector binding and activation. I predicted two major dimer interfaces by employing a powerful prediction algorithm, PRISM (PRotein Interactions by Structural Matching). The first, highly populated β-sheet dimer interface is at the Switch I and effector binding regions. This interface has to be inhibitory to such effectors. The second helical interface may promote Raf‘s activation. To validate the predictions, I mutated some of the residues at the dimer interfaces and investigate the effects of these mutations on Ras dimerization and signaling. The results indicate that K101D/R102E double mutation on the helical interface interferes with Ras dimerization, but does not affect signaling significantly. On the other hand, R41E/K42D double mutation on the beta interface attenuates MAPK signaling, most probably due to the significant decrease in Ras-Raf interaction. To signal, Ras isoforms must be enriched at the plasma membrane (PM). It was suggested that phosphodiesterase-δ (PDEδ) can bind and shuttle some farnesylated Ras isoforms to the PM – but not all. To study Ras/PDEδ interactions, I modeled and simulated the K-Ras4B/PDEδ and N-Ras or K-Ras4A HVR/PDEδ complexes. Earlier data suggested that PDEδ extracts K-Ras4B and N-Ras from the PM; but surprisingly not K-Ras4A. Our results suggest that PDEδ can bind to farnesylated K-Ras4A and N-Ras like K-Ras4B – albeit not as strongly. This weaker binding, coupled with stronger anchoring of K-Ras4A in the membrane, can explain the observation of why PDEδ is unable to effectively extract K-Ras4A.