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.
Lee
F S
FS
Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, MA 02115.
Vallee
B L
BL
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