FUNCTIONAL NUCLEOTIDE-BINDING DOMAIN IN THE F(1)F(0)-ATPSYNTHASE ALPHA-SUBUNIT FROM THE YEAST SCHIZOSACCHAROMYCES-POMBE

The segment R165-T330 of the alpha subunit of Schizosaccharomyces pomb e F1-ATPase, corresponding to a putative nucleotide-binding domain by comparison with related nucleotide-binding proteins, has been overexpr essed in Escherichia coli. Produced as a nonsoluble material, it was p urified in a nonnative form, using a rapid procedure that includes one reversed-phase chromatography step. Refolding of the domain, called D Nalpha19, was achieved quantitatively by using a high-dilution step an d monitored by circular dichroism and intrinsic fluorescence. Once fol ded, DNalpha19 was highly soluble and stable. It bound 1 mol/mol eithe r of adenine or guanine di- or triphosphate nucleotide, with a K(d) ra nging from 2.3 to 5.4 muM, or of methylanthraniloyl derivatives of the same nucleotides, with a K(d) ranging from 0.2 to 0.6 muM. Interestin gly, DNalpha19 was able to hydrolyze nucleoside triphosphates at a low but significant rate. The distance between one tryptophan residue loc ated in the nucleotide-binding site and the ribose-linked methylanthra niloyl group of di- or triphosphate nucleotides was estimated by fluor escence resonance energy transfer to be 13 or 11 angstrom, respectivel y, suggesting that the tryptophan is close to the polyphosphate moiety of the nucleotide. This tryptophan residue was tentatively assigned t o W190 by a hydrophobic cluster comparison with the H-ras p21 protein, suggesting that the putative loop of DNalpha19 containing W190 could play a functional role in nucleotide binding.