Mechanism of free radical nitric oxide-mediated Ras guanine nucleotide dissociation.
Ras proteins cycle between GDP-bound and GTP-bound states to modulate a diverse array of cellular growth processes. In this study, we have elucidated a mechanism by which nitric oxide, in the presence of oxygen (NO/O2), regulates Ras activity. We show that treatment of Ras with NO/O2 causes conversion of Ras-bound GDP into a free 463.3 Da nucleotide-nitration product. Mass and UV/visible spectroscopic analyses suggest that this nitration product is 5-guanidino-4-nitroimidazole diphosphate (NIm-DP), a degradation product of 5-nitro-GDP. These results indicate that NO/O2 mediates Ras guanine nucleotide exchange (GNE) by conversion of Ras-bound GDP into an unstable 5-nitro-GDP. 5-Nitro-GDP can be produced by radical-based reaction of the GDP guanine base with nitrogen dioxide (*NO2). We also provide evidence that the Ras Phe28 side-chain plays a key role in the formation of a NO/O2-induced Ras 5-nitro-GDP product. We previously proposed a mechanism of NO/O2-mediated Ras GNE, in which *NO2, formed by the reaction of NO with O2, generates a Ras Cys118 thiyl radical (Ras-S118) intermediate. In the present study, we provide evidence for a radical-based mechanism of NO/O2-mediated Ras GNE. According to this mechanism, reaction of NO with O2 produces *NO2. *NO2 then reacts with Ras to produce Ras-S118, which withdraws an electron from the Ras-bound guanine nucleotide base to produce a guanine nucleotide diphosphate cation radical (G(+)-DP) via the Phe28 side-chain. G(+)-DP is subsequently converted to a neutral radical, and can react with another *NO2 to produce 5-nitro-GDP. This radical-based reaction process disrupts key binding interactions between Ras and the guanine base, resulting in release of GDP from Ras and its conversion to free 5-nitro-GDP. This mechanism is likely to be common to other NKCD motif-containing Ras superfamily GTPases, as NO/O2 also facilitates GNE on the redox-active Rap1A and Rab3A GTPases.
Heo
Jongyun
J
Department of Biochemistry and Biophysics, The University of North Carolina, 530 Mary Ellen Jones Building, Chapel Hill, NC 27599-7260, USA.
Prutzman
Kirk C
KC
Mocanu
Viorel
V
Campbell
Sharon L
SL
D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D005609:Free Radicals / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D009569:Nitric Oxide / Q000378:metabolism*; D009585:Nitrogen Dioxide / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000378:metabolism*; D020663:ras Guanine Nucleotide Exchange Factors / Q000378:metabolism*
D005609:Free Radicals; D020663:ras Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate; D009569:Nitric Oxide; D006160:Guanosine Triphosphate; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D009585:Nitrogen Dioxide; D010100:Oxygen