Functional and structural interactions of the Rab5 D136N mutant with xanthine nucleotides.
Rab5 is a Ras-related GTPase which regulates endosomal fusion. The D136N mutant of Rab5, which was predicted to switch specificity from guanine to xanthine nucleotides, was expressed in E. coli, extracted with urea, purified by column chromatography, and refolded by stepwise dialysis against buffer containing XDP. The purified protein bound xanthine nucleotides with considerably higher affinity than guanine nucleotides. In vitro prenylation of the mutant protein was highly dependent on xanthosine diphosphate. In contrast, both the wild type and mutant proteins were protected from proteolysis equally well by non-cognate and cognate triphosphate nucleosides at high concentration. The D136N Rab5 mutant appears to be a valuable reagent in conjunction with xanthine nucleotides for the study of protein-nucleotide interactions in systems in which multiple GTPases are active, although interactions with non-cognate nucleotides should be evaluated if they are present at high concentration.
Hoffenberg
S
S
Baylor College of Medicine, Department of Medicine, Houston, TX 77030, USA.
Nikolova
L
L
Pan
J Y
JY
Daniel
D S
DS
Wessling-Resnick
M
M
Knoll
B J
BJ
Dickey
B F
BF
D001483:Base Sequence; D004926:Escherichia coli; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009711:Nucleotides / Q000378:metabolism*; D017368:Protein Prenylation; D011994:Recombinant Proteins / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D019820:Xanthine; D014970:Xanthines / Q000378:metabolism*; D020696:rab5 GTP-Binding Proteins
D009711:Nucleotides; D011994:Recombinant Proteins; D012265:Ribonucleotides; D014970:Xanthines; D019820:Xanthine; C065326:xanthosine 5'-triphosphate; D006160:Guanosine Triphosphate; D014357:Trypsin; D019204:GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins