Modular mutagenesis of human placental ribonuclease inhibitor, a protein with leucine-rich repeats.
Human placental ribonuclease inhibitor (PRI) is a potent protein inhibitor of pancreatic ribonucleases and the homologous blood vessel-inducing protein angiogenin. Although inhibition by PRI occurs with a 1:1 stoichiometry, its primary structure is composed predominantly of seven internal leucine-rich repeats. These internal repeats were systematically deleted either singly or in combination by "modular" mutagenesis. Deletion of repeat units 3 plus 4 or repeat unit 6 results in mutants that both bind to and inhibit ribonuclease A. Therefore, the angiogenin/ribonuclease binding site in PRI must reside primarily or entirely in repeats 1, 2, 5, or 7, the short N- or C-terminal segments, or a combination of these. Deletion of repeat units 3-5, 5-6, or 5 alone results in mutants that exhibit only binding activity. Hence, the binding site cannot reside exclusively in repeat 5. Other internal deletions or N- or C-terminal deletions of 6-86% of the protein all abolish activity. These results suggest that PRI has a modular structure, with one primary structural repeat constituting one module. The approach taken may be applicable to other proteins with repeat structures.
Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, MA 02115.
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D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D006801:Humans; D007700:Kinetics; D007930:Leucine; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010926:Placental Hormones / Q000235:genetics*; D010957:Plasmids; D011247:Pregnancy; D012091:Repetitive Sequences, Nucleic Acid; D012259:Ribonuclease, Pancreatic / Q000037:antagonists & inhibitors / Q000378:metabolism; D012260:Ribonucleases / Q000037:antagonists & inhibitors
D015345:Oligonucleotide Probes; D010926:Placental Hormones; C022052:placental ribonuclease inhibitor; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic; D007930:Leucine