Control of Small GTPase Ras Using a Calmodulin-based Ionochromic Nanodevice

Ziyun Zhang, Yassine Sabek, and Shinsaku Maruta^*

Division of Bioinformatics, Graduate School of Engineering, Soka University, Hachioji, Tokyo, Japan

Corresponding Author E-mail: maruta@soka.ac.jp

ABSTRACT: The small GTP-binding protein, HRas, is a switch-like molecule that plays an important role in the regulation of many cell processes. It is activated by binding to GTP and is inactivated when GTP is hydrolyzed to GDP. Ras has two accessory factors, guanine accelerate protein (GAP) and guanine nucleotide exchange factor (GEF), which facilitate its switching function by accelerating GTP hydrolysis and GDP/GTP exchange. Calmodulin (CaM) is a crucial signaling and regulatory molecule involved in many calcium-dependent processes. In the calcium-bound state, CaM binds tightly to the M13 peptide and IQ motif. Because there are no reports using CaM as an ionochromic switch system, CaM was used here to artificially control Ras. An HRas fusion protein with M13 (M13-HRas) was expressed using an established Escherichia coli expression system. M13-HRas showed 73% ion-regulation when the regulatory factors GAP and GEF were present. The CaM-bound state inhibited the interaction between M13-HRas and GST-Raf while maintaining a similar GTPase activity regulation ratio. Finally, the inhibition of CaM binding between M13-HRas and two accessory factors was confirmed. Thus, modifying the G protein functional site with M13 enabled ionochromic control of G protein function with CaM, which has implications for cancer therapy.

KEYWORDS: Calmodulin; Ionochromic; Ras; Small Gtpase

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