The role of the conserved switch II glutamate in guanine nucleotide exchange factor-mediated nucleotide exchange of GTP-binding proteins.
Guanine nucleotide exchange factors (GEFs) regulate the activity of small G proteins by catalysing the intrinsically slow exchange of GDP for GTP. The mechanism involves the formation of trimeric G protein-nucleotide-GEF complexes, followed by the release of nucleotide to form stable binary G protein-GEF complexes. A number of structural studies of G protein-GEF complexes have shown large structural changes induced in the nucleotide binding site. Together with a recent structure of a trimeric complex, these studies have suggested not only some common principles but also large differences in detail in the GEF-mediated exchange reaction. Several structures suggested that a glutamic acid residue in switch II, which is part of the DxxGQE motif and highly conserved in Ras-like G proteins, might have a decisive mechanistic role in GEF-mediated nucleotide exchange reactions. Here we show that mutation of the switch II glutamate to Ala severely impairs GEF-catalysed nucleotide exchange in most, but not all, Ras family G proteins, explaining its high sequence conservation. The residue determines the initial approach of GEF to the nucleotide-loaded G protein and does not appreciably affect the formation of a binary nucleotide-free complex. Its major effect thus appears to be the removal of the P-loop lysine from its interaction with the nucleotide.
Gasper
Raphael
R
Abteilung Strukturelle Biologie, Max-Planck-Institut für Molekulare Physiologie, 44227 Dortmund, Germany.
Thomas
Christoph
C
Ahmadian
Mohammad Reza
MR
Wittinghofer
Alfred
A
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D002384:Catalysis; D017124:Conserved Sequence; D018698:Glutamic Acid / Q000737:chemistry*; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D020559:Monomeric GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D009154:Mutation; D020663:ras Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018631:ras Proteins / Q000737:chemistry / Q000378:metabolism
D020662:Guanine Nucleotide Exchange Factors; D020663:ras Guanine Nucleotide Exchange Factors; D018698:Glutamic Acid; D020559:Monomeric GTP-Binding Proteins; D018631:ras Proteins; D000409:Alanine