Guanine nucleotide binding properties of the mammalian RalA protein produced in Escherichia coli.
The simian ralA cDNA was inserted in a ptac expression vector, and high amounts of soluble ral protein were expressed in Escherichia coli. The purified p24ral contains 1 mol of bound nucleotide/mol of protein that can be exchanged against external nucleotide. The ral protein exchanges GDP with a t 1/2 of 90 min at 37 degrees C in the presence of Mg2+, and has a low GTPase activity (0.07 min-1 at 37 degrees C). We have also studied its affinity for various guanine nucleotides and analogs. NMR measurements show that the three-dimensional environment around the nucleotide is similar in p21ras and p24ral. In addition to these studies on the wild-type ral protein, we used in vitro mutagenesis to introduce substitutions corresponding to the Val12, Val12 + Thr59, and Leu61 substitutions of p21ras. These mutant ral proteins display altered nucleotide exchange kinetics and GTPase activities, however, the effects of the substitutions are less pronounced than in the ras proteins. p24ralVal12 + Thr59 autophosphorylates on the substituted Thr, as a side reaction of the GTP hydrolysis, but the rate is much lower than those of the Thr59 mutants of p21ras. These results show that ras and ral proteins have similar structures and biochemical properties. Significant differences are found, however, in the contribution of the Mg2+ ion to GDP binding, in the rate of the GTPase reaction and in the sensitivity of these two proteins to substitutions around the phosphate-binding site, suggesting that the various "small G-proteins" of the ras family perform different functions.
Frech
M
M
Max-Planck-Institut für medizinische Forschung, Abteilung Biophysik, Heidelberg, Federal Republic of Germany.
Schlichting
I
I
Wittinghofer
A
A
Chardin
P
P
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005822:Genetic Vectors; D006150:Guanine Nucleotides / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D015689:Oncogene Protein p21(ras) / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D006150:Guanine Nucleotides; D015345:Oligonucleotide Probes; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D015636:Magnesium Chloride; D006153:Guanosine Diphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D015689:Oncogene Protein p21(ras)