Mapping of ATP binding regions in poly(A) polymerases by photoaffinity labeling and by mutational analysis identifies a domain conserved in many nucleotidyltransferases

• Published in: Protein science Vol. 8; no. 11; pp. 2380 - 2391
• Main Authors: MARTIN, GEORGES, JENÖ, PAUL, KELLER, WALTER
• Format: Journal Article
• Language: English
• Published: Bristol Cambridge University Press 01.11.1999; Cold Spring Harbor Laboratory Press
• Subjects: 3′‐ends; 8‐azido‐ATP; Adenosine Triphosphate - analogs & derivatives; Adenosine Triphosphate - metabolism; Adenosine Triphosphate - pharmacokinetics; Affinity Labels; Amino Acid Sequence; Amino Acid Substitution; Animals; Azides - pharmacokinetics; Binding Sites; Cattle; Conserved Sequence; helical turn motif; Helix-Turn-Helix Motifs; Kinetics; Models, Molecular; Molecular Sequence Data; mRNA; Mutagenesis, Site-Directed; nucleotidyltransferase; Nucleotidyltransferases - chemistry; Nucleotidyltransferases - metabolism; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Peptide Mapping; Polynucleotide Adenylyltransferase - chemistry; Polynucleotide Adenylyltransferase - metabolism; Protein Conformation; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Schizosaccharomyces - enzymology; Sequence Alignment; Sequence Homology, Amino Acid; UV cross‐linking; UV cross-linking; nucleotidyltransferase; mRNA; 3′-ends; helical turn motif; 8-azido-ATP
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1110/ps.8.11.2380

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.