Modular mutagenesis of human placental ribonuclease inhibitor, a protein with leucine-rich repeats.

Abstract

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.