Study Illuminates K-Ras4B Activation, Which May Help Predict Drug Resistance

By Chris Worthington, Staff Writer; photo by Richard Frederickson, Staff Photographer; contributed images
Hyunbum Jang, Ph.D., and Ruth Nussinov, Ph.D.

Ruth Nussinov, Ph.D. (right), senior investigator, Cancer and Inflammation Program and head of Computational Structural Biology section, Center for Cancer Research, with Hyunbum Jang, Ph.D., senior computational scientist.

Editor's note: Platinum Highlight articles are noteworthy publications selected periodically by Craig Reynolds, Ph.D., associate director, NCI at Frederick, from among the most recently published Platinum Publications.

Until recently, researchers studying RAS, a family of proteins involved in transmitting signals within cells, believed that the exchange of guanosine 5’-diphosphate (GDP) by guanosine triphosphate (GTP) was sufficient to activate the protein. Once activated, RAS can cause unintended and overactive signaling in cells, which can lead to cell division and, ultimately, cancer.

However, as reported in a FASEB Journal paper, the GDP/GTP exchange may not be sufficient for activation. Instead, the results published by Hyunbum Jang, Ph.D., Ruth Nussinov, Ph.D., and their colleagues suggest that a number of factors, including the GDP/GTP exchange, hypervariable region sequestration, farnesyl insertion, and orientation/localization of the catalytic domain at the membrane conjointly determine the active or inactive state of K-Ras4B, the member of the RAS family on which the study focused.

“K-Ras4B is the most oncogenic isoform of the RAS family,” Nussinov said, meaning it is the most active contributor to tumor formation. “Clarifying the mechanism of K-Ras4B activation and identifying the active conformation are important goals and will be useful in rational drug discovery.”

According to Nussinov, the team chose to study RAS activation because the molecular biophysics of the protein—its environment and signaling, from a structural standpoint—can help explain the Ras mechanism in oncogenic cells and help predict drug resistance.

Previously, the team had published a semisynthetic method for production of farnesylated and methylated K-Ras4B, and a modified version of that method was used in the research discussed here. To obtain structural details of the farnesylated K-Ras4B, the team performed molecular dynamics simulations on K-Ras4B both in aqueous and lipid environments. Their observations led them to conclude that GTP binding alone does not determine an active conformation.

Jang, the first author on the paper, said “This is the first computational effort to model the full-length K-Ras4B protein with the post-translational modifications interacting with the PS-rich membrane.”

The research group completed the computational part of the study at the NCI at Frederick campus using the Biowulf high-performance computing system. The experimental nuclear magnetic resonance study was performed in the University of Illinois in Chicago.

Nussinov has worked at the NCI at Frederick since 1985, while Jang has worked there since 2005.

Snapshots representing structures of K-Ras4B proteins at the anionic lipid bilayer composed of DOPC and DOPS lipids. Cartoons for the catalytic domains are shown in green and pink for the GDP-bound and GTP-bound states, respectively. The hypervariable region in the tube representation is colored blue, and the farnesyl as a stick is colored yellow. In the catalytic domain, the red sticks and green spheres represent the nucleotide and magnesium ion, respectively. For the lipid bilayer, white surface denotes DCartoons representing the inactive and active states of K-Ras4B at the anionic membrane. The balloon represents the K-Ras4B catalytic domain, the blue thread tied to the balloon represents the hypervariable region (HVR), and the yellow sawtooth represents the farnesyl. In the inactive state, the membrane-interacting HVR hauls the effector lobe to the membrane surface, burying the effector binding site. In the active state, the catalytic domain liberates the HVR, exposing the effector binding site and fluctu