Influence of Mg2+ on the structure and function of Rab5.
Mg2+ inhibits GDP release from Rab5WT but not from Rab5S34N, a mutant lacking Ser34 critical for Mg2+ coordination in the nucleotide binding pocket. Thus, inhibition of GDP release is apparently exerted via coordination of Mg2+ between Rab5 and GDP. Mg2+ also induces conformational changes in Rab5WT, demonstrated by increased tryptophan fluorescence intensity and a red shift in lambda max for the GDP-bound protein. Mg(2+)-induced fluorescence changes are not observed for Rab5S34N. The correlation between Mg2+ effects on nucleotide exchange and the fluorescence properties of Rab5 suggests that a conformation promoted through Mg2+ coordination with Ser34 also contributes to inhibition of GDP release. The role of structural changes in GDP release was investigated using C- and N-terminal truncation mutants. Similar to Rab5WT, Mg2+ inhibits GDP release and alters the fluorescence of Rab5(1-198) but only partially inhibits release from Rab5(23-198) and fails to induce changes in the latter's fluorescence properties. Since Rab5(23-198) maintains Ser34 necessary for Mg2+ coordination, the lack of Mg(2+)-induced fluorescence changes suggests a requirement for the N-terminal domain to promote a conformation blocking GDP release. A model for mechanisms of interaction between Ras-like proteins and their exchange factors is proposed.
Pan
J Y
JY
Department of Nutrition, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
Sanford
J C
JC
Wessling-Resnick
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence; D014357:Trypsin; D014364:Tryptophan / Q000032:analysis; D020696:rab5 GTP-Binding Proteins
D017931:DNA Primers; D010446:Peptide Fragments; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D014364:Tryptophan; D014357:Trypsin; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; D008274:Magnesium