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pH-dependent perturbation of Ras-guanine nucleotide interactions and Ras guanine nucleotide exchange

Jongyun Heo1, Guanghua Gao, Sharon L Campbell

  • 1Department of Biochemistry and Biophysics, University of North Carolina, 530 Mary Ellen Jones Building, Chapel Hill, North Carolina 27599-7260, USA.

Biochemistry
|August 4, 2004
PubMed

Insights

Investigating pH effects on Ras proteins reveals critical Mg2+ interactions influencing guanine nucleotide binding and exchange. This study clarifies Ras regulation by pH and nitric oxide, impacting cell signaling pathways.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Signaling

Background:

  • p21Ras proteins regulate crucial cellular processes by cycling between active (GTP-bound) and inactive (GDP-bound) states.
  • Cellular factors like guanine nucleotide exchange factors (GEFs) and nitric oxide (NO) modulate Ras activity.
  • Understanding Ras-guanine nucleotide interactions is key to deciphering signal transduction.

Purpose of the Study:

  • To investigate the pH dependence of Ras-GDP interactions and Ras-guanine nucleotide exchange (GNE).
  • To elucidate the role of pH and Mg2+ in modulating Ras-guanine nucleotide binding affinity.
  • To clarify the mechanism of nitric oxide-mediated Ras GNE.

Main Methods:

  • Nuclear Magnetic Resonance (NMR) spectroscopy to identify pH-sensitive residues.
  • Kinetic studies to analyze Ras-guanine nucleotide exchange rates.
  • Analysis of Mg2+ and guanine nucleotide binding affinities under varying pH conditions.

Main Results:

  • pH-sensitive amide protons were identified in Ras switch I and switch II regions.
  • Mg2+ interacting residues show pH-sensitive shifts coupled to Mg2+ and nucleotide binding.
  • Perturbation of Mg2+ interactions is critical for pH-dependent nucleotide dissociation from Ras.
  • These regions undergo conformational changes upon GEF (SOS) binding.
  • The H+ byproduct of NO reaction is unlikely to mediate NO-induced Ras GNE.

Conclusions:

  • pH significantly influences Ras-guanine nucleotide binding and exchange through Mg2+ interactions.
  • Ras conformational changes in switch regions are crucial for GEF-mediated regulation.
  • Nitric oxide-mediated Ras GNE does not rely on the H+ byproduct, suggesting a different mechanism.

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