Distinct association of HRAS and KRAS with Mn²⁺ ion illustrated by paramagnetic NMR

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Rat sarcoma virus oncogene (RAS) proteins are critical oncogenic proteins involved in essential intracellular signaling pathways.
• RAS proteins possess a primary Mg²⁺ binding site crucial for active site stability.
• A secondary metal ion binding site has been identified in HRAS, previously shown to bind Ca²⁺.

Purpose of the Study:

• To investigate the interaction of manganese (Mn²⁺) with Harvey RAS homolog (HRAS) and Kirsten RAS homolog (KRAS) proteins.
• To elucidate the role of Mn²⁺ in the second metal ion binding site and its influence on RAS protein conformation.

Main Methods:

• High-resolution Nuclear Magnetic Resonance (NMR) spectroscopy was employed to study Mn²⁺ interactions.
• Paramagnetic NMR techniques were utilized to probe conformational changes and binding sites.

Main Results:

• Mn²⁺ was found to bind to both the second metal ion binding site and the switch I and II regions in HRAS and KRAS.
• Paramagnetic NMR data revealed distinct conformational differences in helix α3 and the adjacent loop between HRAS and KRAS.
• These conformational variations are associated with Mn²⁺ binding, suggesting a role in modulating RAS protein function.

Conclusions:

• Mn²⁺ interacts with HRAS and KRAS at a secondary binding site and influences key functional regions.
• The study highlights Mn²⁺ as a potential physiological ion for the secondary binding site, impacting RAS signaling.
• Identified conformational differences between HRAS and KRAS provide insights into their specific biological roles and regulation.