RalGDS-Ral sinyal yolağının Moleküler Dinamik Simulasyon metodu ile incelenmesi

Ras family is composed of at least 35 proteins that are known as GTP-GDP mediated molecular switches that regulate most of the cellular processes through physical interactions with their wide variety of effector proteins. In response to the diverse extracellular stimuli, they become activated, and then recruit their effector proteins (Raf, PI3K, and RalGDS) to the plasma membrane and transmit the signal. Raf cascade and PI3K pathways are well-established effector pathways; RalGDS lately become the research scrutiny. There are four prominent members of the family: H-Ras, N-Ras, K-Ras4A and K-Ras4B proteins that are found as the most frequently mutated proteins in human cancer types. The other well-known Ras members are Rap1 and Ral proteins. Whereas prominent members are well-characterized; the cellular functions of Rap1 and Ral are yet to be understood. In this study, the main aim is to elaborate the RalGDS-Ral signalling pathway in complex with Ras family members (K-Ras, Rap1) and Ral effector proteins using molecular dynamics simulations. The five Ras systems and two Ral systems were generated to elaborate upstream and downstream of RalGDS signaling cascade. The five Ras systems are comprised of K-Ras4Bwt-RalGDS-RBD as a wild-type system, K-Ras4BG12D-RalGDS-RBD and K-Ras4BG12V-RalGDS-RBD as control systems, K-Ras4BG12V+E37G-RalGDS-RBD as a mutational system, and Rap1wt-RalGDS-RBD as a Rap1 system; the two Ral sytems are composed of Ral-Sec5 and Ral-Exo84 effector interactions. The systems were designed to give insight into details of mechanism behind Ras-induced RalGDS-Ral pathway. The frequently involved residues that are responsible for stable complex formations and strong bond formations were investigated via MD simulations. We proposed the residues, Ser39 on Ras; Lys31 in Rap1; Tyr31, Asn54 and Asp56 on RalGDS as significant residues that amplify binding affinity between Ras family members and Ras Binding Domain of RalGDS. It is known that Sec5 and Exo84 have partially overlapping residues on RalA. We discovered most of the common involved residues on RalA in both interactions, which are Glu44, Ala48, Asp49, Ser50, Arg52, Tyr75. The comparison of two Ral systems allowed us to identify important residues that amplify binding affinity of RalA to its effector proteins. These residues are Asp49, Glu73, Asp74, and Asn81. We further demonstrate the frequently involves important residues on Sec5 and Exo84. Ras proteins have been scrutinizing for decades; however, they are still undruggable. The long-term purpose of this intense research is to develop small molecules that target Ras dependent RalGDS signaling particularly.