Conformational states of the small G protein Arf-1 in complex with the guanine nucleotide exchange factor ARNO-Sec7.
Arf1 is a small G protein involved in vesicular trafficking, and although it is only distantly related to Ras, it adopts a similar three-dimensional structure. In the present work, we study Arf1 bound to GDP and GTP and its interactions with one of its guanosine nucleotide exchange factors, ARNO-Sec7. The (31)P NMR spectra of Arf1.GDP.Mg(2+) and Arf1.GTP.Mg(2+) share the general features typical for all small G proteins studied so far. Especially, the beta-phosphate resonances of the bound nucleotide are shifted strongly downfield compared with the resonance positions of the free magnesium complexes of GDP and GTP. However, no evidence for an equilibrium between two conformational states of Arf1.GDP.Mg(2+) or Arf1.GTP.Mg(2+) could be observed as it was described earlier for Ras and Ran. Glu(156) of ARNO-Sec7 has been suggested to play as "glutamic acid finger" an important role in the nucleotide exchange mechanism. In the millimolar concentration range used in the NMR experiments, wild type ARNO-Sec7 and ARNO-Sec7(E156D) do weakly interact with Arf1.GDP.Mg(2+) but do not form a strong complex with magnesium-free Arf1.GDP. Only wild type ARNO-Sec7 competes weakly with GDP on Arf1.GDP.Mg(2+) and leads to a release of GDP when added to the solution. The catalytically inactive mutants ARNO-Sec7(E156A) and ARNO-Sec7(E156K) induce a release of magnesium from Arf1.GDP.Mg(2+) but do not promote GDP release. In addition, ARNO-Sec7 does not interact or only very weakly interacts with the GTP-bound form of Arf1, opposite to the observation made earlier for Ran, where the nucleotide exchange factor RCC1 forms a complex with Ran.GTP.Mg(2+) and is able to displace the bound GTP.
Kremer
Werner
W
Institut für Biophysik und physikalische Biochemie, Universität Regensburg, D-93040 Regensburg, Germany.
Steiner
Guido
G
Béraud-Dufour
Sophie
S
Kalbitzer
Hans Robert
HR
D020823:ADP-Ribosylation Factor 1 / Q000737:chemistry*; D002384:Catalysis; D020134:Catalytic Domain; D004926:Escherichia coli / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000737:chemistry* / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000737:chemistry; D007477:Ions; D008274:Magnesium / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D009711:Nucleotides / Q000737:chemistry; D010758:Phosphorus / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation
D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D007477:Ions; D009711:Nucleotides; C066755:Sec7 guanine nucleotide exchange factors; C102474:cytohesin-2; D006153:Guanosine Diphosphate; D010758:Phosphorus; D018698:Glutamic Acid; D006160:Guanosine Triphosphate; D020823:ADP-Ribosylation Factor 1; D008274:Magnesium