Nucleotide-Specific Autoinhibition of Full-Length K-Ras4B Identified by Extensive Conformational Sampling

Balint Dudas,Erika Balog,Hyunbum Jang,Franci Merzel,David Perahia,Ruth Nussinov

doi:10.3389/fmolb.2020.00145

Abstract

K-Ras is one of the most frequently mutated oncogenes in human tumor cells. It consists of a well-conserved globular catalytic domain and a flexible tail-like hypervariable region (HVR) at its C-terminal end. It plays a key role in signaling networks in proliferation, differentiation, and survival, undergoing a conformational switch between the active and inactive states. It is regulated through the GDP-GTP cycle of the inactive GDP-bound and active GTP-bound states. Here, without imposing any prior constraints, we mapped the interaction pattern between the catalytic domain and the HVR using Molecular Dynamics with excited Normal Modes (MDeNM) starting from an initially extended HVR conformation for both states. Our sampling captured similar interaction patterns in both GDP- and GTP-bound states with shifted populations depending on the bound nucleotide. In the GDP-bound state, the conformations where the HVR interacts with the effector lobe are more populated than in the GTP-bound state, forming a buried thus autoinhibited catalytic site; in the GTP-bound state conformations where the HVR interacts with the allosteric lobe are more populated, overlapping the α3/α4 dimerization interface. The interaction of the GTP with Switch I and Switch II is stronger than that of the GDP in line with a decrease in the fluctuation upon GTP binding.

Highlights

In order to identify the interacting residues of the catalytic domain and the hypervariable region (HVR), a distance-based criterion was applied to the conformations generated by Molecular Dynamics with excited Normal Modes (MDeNM): if the distance between two heavy atoms of a residue within the catalytic domain and a residue in the HVR was less than 5.5 Å, these two residues were considered interacting (Bowerman and Wereszczynski, 2016)
Our analysis shows that the interacting catalytic domain-HVR residues detected by MDeNM contain all the interacting residues identified by nuclear magnetic resonance (NMR) measurements (Thapar et al, 2004; Chavan et al, 2015; Lu et al, 2015; Jang et al, 2016a)
As MDeNM revealed and was confirmed by molecular dynamics (MD) simulations, almost all catalytic domain-HVR interactions exist in both GDP- and GTP-bound K-Ras4B

Summary

Introduction

Ras signaling is determined by the GTPase cycle: inactive GDP-bound and active GTP-bound states, which change the conformation of Ras and its affinity to bind to downstream effectors— such as Raf kinase (Pacold et al, 2000; Fetics et al, 2015) and phosphatidylinositol 3-kinase (PI3K) (Pacold et al, 2000). Guanine nucleotide exchange factors (GEFs) catalyze the release of GDP which is followed by GTP binding, and the GTPase activating. MDeNM: Autoinhibition of K-Ras4B proteins (GAPs) catalyze GTP hydrolysis (Bos et al, 2007), resulting in GDP-bound conformations. Oncogenic mutations lock Ras in its active, GTP-bound conformation, being always available to downstream effectors, which leads to uncontrolled cell growth and cancer (Prior et al, 2012). Mutant GDP-bound proteins may shift their conformations to a GTP-boundlike state

Methods

Results

Conclusion