Related Experiment Video
Updated: May 6, 2026
RhoC GTPase Activation Assay
Published on: August 22, 2010
DRoP: a water analysis program identifies Ras-GTP-specific pathway of communication between membrane-interacting
Bradley M Kearney1, Christian W Johnson2, Daniel M Roberts3
1Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA; Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695, USA.
Abstract:
Ras GTPase mediates several cellular signal transduction pathways and is found mutated in a large number of cancers. It is active in the GTP-bound state, where it interacts with effector proteins, and at rest in the GDP-bound state. The catalytic domain is tethered to the membrane, with which it interacts in a nucleotide-dependent manner. Here we present the program Detection of Related Solvent Positions (DRoP) for crystallographic water analysis on protein surfaces and use it to study Ras. DRoP reads and superimposes multiple Protein Data Bank coordinates, transfers symmetry-related water molecules to the position closest to the protein surface, and ranks the waters according to how well conserved and tightly clustered they are in the set of structures. Coloring according to this rank allows visualization of the results. The effector-binding region of Ras is hydrated with highly conserved water molecules at the interface between the P-loop, switch I, and switch II, as well as at the Raf-RBD binding pocket. Furthermore, we discovered a new conserved water-mediated H-bonding network present in Ras-GTP, but not in Ras-GDP, that links the nucleotide sensor residues R161 and R164 on helix 5 to the active site. The double mutant RasN85A/N86A, where the final link between helix 5 and the nucleotide is not possible, is a severely impaired enzyme, while the single mutant RasN86A, with partial connection to the active site, has a wild-type hydrolysis rate. DRoP was instrumental in determining the water-mediated connectivity networks that link two lobes of the catalytic domain in Ras.
Insights
We developed DRoP, a program for analyzing water molecules on protein surfaces, and used it to study Ras GTPase. DRoP identified conserved water networks crucial for Ras function and cancer-related mutations.
Area of Science:
- Structural Biology
- Biochemistry
- Computational Biology
Background:
- Ras GTPase is a key regulator of cellular signaling, frequently mutated in cancers.
- Ras activity is controlled by its nucleotide-bound state (GTP or GDP) and membrane tethering.
- Understanding Ras-protein interactions requires detailed analysis of its surface hydration.
Purpose of the Study:
- To introduce Detection of Related Solvent Positions (DRoP), a novel program for crystallographic water analysis.
- To investigate the role of water molecules in Ras GTPase structure and function using DRoP.
- To identify conserved water-mediated networks involved in Ras effector binding and nucleotide sensing.
Main Methods:
- Development and application of the DRoP program for analyzing multiple Protein Data Bank structures.
- DRoP identifies, superimposes, and ranks conserved water molecules on protein surfaces.
- Analysis of wild-type and mutant Ras structures (Ras-GTP and Ras-GDP) to map water networks.
Main Results:
- DRoP identified highly conserved water molecules at the Ras effector-binding interface (P-loop, switch I, switch II) and Raf-RBD pocket.
- A novel water-mediated hydrogen-bonding network was discovered in Ras-GTP, linking nucleotide sensor residues to the active site.
- Mutational analysis (RasN85A/N86A, RasN86A) confirmed the importance of this water network for Ras enzyme activity.
Conclusions:
- DRoP is an effective tool for visualizing and analyzing conserved water networks on protein surfaces.
- Conserved water molecules play critical roles in Ras effector recognition and catalytic activity.
- Water-mediated interactions are essential for linking Ras's nucleotide-binding site to its catalytic machinery.
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