Chemical and Biological Engineering PhD Thesis by E. Sıla Özdemir

KOÇ UNIVERSITY

GRADUATE SCHOOL OF SCIENCES & ENGINEERING

CHEMICAL AND BIOLOGICAL ENGINEERING

PhD THESIS DEFENSE BY E. SILA ÖZDEMİR

Title: Understanding Rho GTPase Interactions with Scaffolding Proteins and Ras Protein Shuttling Mechanisms

Speaker: E. Sıla Özdemir

Time: September 3rd, 2018, 10:00

Place: ENG208

Koç University

Rumeli Feneri Yolu

Sariyer, Istanbul

Thesis Committee Members:

Prof. Özlem Keskin (Advisor, Koç University)

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

Prof. Tarık Tihan (Koç University, UCSF)

Assoc. Prof. Seda Keskin (Koç University)

Asst. Prof. Özlem Ulucan (Bilgi University)

Asst. Prof. Mert Gür (Istanbul Technical University)

Abstract:

The Rho GTPase family and the Ras GTPase family are subfamilies of the Ras GTPase superfamily. The GTPases selectively interact with structurally and functionally diverse effectors, such as IQ motif–containing GTPase-activating proteins (IQGAP) and phosphodiesterase-δ (PDEδ). Rho GTPases are distinguished from other GTPases by the presence of an “insert loop,” which is rich in charged residues in a helical motif. The Rho GTPases, Cdc42 and Rac1, in their GTP-bound active forms, interact with all three human IQGAPs.  The IQGAP–Cdc42 interaction promotes metastasis by enhancing actin polymerization. However, despite their high sequence identity, Cdc42 and Rac1 differ in their interactions with IQGAP. The exact mechanism of Cdc42 and Rac1 binding to IQGAP is unclear. Using all atom molecular dynamics (MD) simulations, the detailed mechanisms of Cdc42 and Rac1 interactions with IQGAP2 were studied. It was observed that the Cdc42 “insert loop” is important for the interaction of the first Cdc42 and allows to the binding of the second Cdc42. By contrast, differences in Rac1 insert-loop sequence and structure precluded its interaction with IQGAP and only one Rac1 can bind. Ras GTPase subfamily protein, Ras is highly mutated in human cancers. Proper localization of Ras proteins at the plasma membrane (PM) is crucial for their functions. Their localization is mediated by PDEδ. GTP-bound Arf-like protein 2 (Arl2) is a regulator of PDEδ-mediated transport of farnesylated proteins; however, the exact mechanism of Arl2-assisted release of farnesylated proteins from PDEδ is still unclear. Using MD simulations, detailed mechanisms of Arl2-mediated release of KRas4B, the most abundant oncogenic Ras isoform, from PDEδ were investigated. The detailed analysis showed that allosteric changes (involving β6 of PDEδ and additional PDEδ residues) compress the hydrophobic PDEδ pocket and push the KRas4B out.

Single amino acid variation (SAV) distribution in protein-protein interaction (PPI) interfaces was also investigated. Interfaces have a small subset of residues called hot spots that contribute significantly to the binding energy, and they may form clusters called hot regions. Singlet hot spots are the single amino acid hot spots outside of the hot regions. Statistical and structural analyses of SAVs were performed with literature curated experimental thermodynamics data, and demonstrated that SAVs which destabilize PPIs and cause diseases are more likely to be found in singlet hot spots rather than hot regions and than energetically less important interface residues. This part of the thesis demonstrates that SAVs in singlet hot spot residues have significant effects on protein stability and function.