Mapping of ATP binding regions in poly(A) polymerases by photoaffinity labeling and by mutational analysis identifies a domain conserved in many nucleotidyltransferases.
We have identified regions in poly(A) polymerases that interact with ATP. Conditions were established for efficient cross-linking of recombinant bovine and yeast poly(A) polymerases to 8-azido-ATP. Mn2+ strongly stimulated this reaction due to a 50-fold lower Ki for 8-azido-ATP in the presence of Mn2+. Mutations of the highly conserved Asp residues 113, 115, and 167, critical for metal binding in the catalytic domain of bovine poly(A) polymerase, led to a strong reduction of cross-linking efficiency, and Mn2+ no longer stimulated the reaction. Sites of 8-azido-ATP cross-linking were mapped in different poly(A) polymerases by CNBr-cleavage and analysis of tryptic peptides by mass spectroscopy. The main cross-link in Schizosaccharomyces pombe poly(A) polymerase could be assigned to the peptide DLELSDNNLLK (amino acids 167-177). Database searches with sequences surrounding the cross-link site detected significant homologies to other nucleotidyltransferase families, suggesting a conservation of the nucleotide-binding fold among these families of enzymes. Mutations in the region of the "helical turn motif" (a domain binding the triphosphate moiety of the nucleotide) and in the suspected nucleotide-binding helix of bovine poly(A) polymerase impaired ATP binding and catalysis. The results indicate that ATP is bound in part by the helical turn motif and in part by a region that may be a structural analog to the fingers domain found in many polymerases.
Martin
G
G
Department of Cell Biology, Biozentrum, University of Basel, Switzerland.
Jenö
P
P
Keller
W
W
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism* / Q000493:pharmacokinetics; D000345:Affinity Labels; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001386:Azides / Q000493:pharmacokinetics; D001665:Binding Sites; D002417:Cattle; D017124:Conserved Sequence; D019077:Helix-Turn-Helix Motifs; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D011062:Polynucleotide Adenylyltransferase / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D000345:Affinity Labels; D001386:Azides; D010446:Peptide Fragments; D011994:Recombinant Proteins; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D009713:Nucleotidyltransferases; D011062:Polynucleotide Adenylyltransferase