J. Mol. Biol. (1996) 264, 1028–1043 Mechanism of Ribonuclease Inhibition by Ribonuclease Inhibitor Protein Based on the Crystal Structure of its Complex with Ribonuclease A Bostjan Kobe 1,2 * and Johann Deisenhofer 2 We describe the mechanism of ribonuclease inhibition by ribonuclease 1 St. Vincent’s Institute of Medical Research inhibitor, a protein built of leucine-rich repeats, based on the crystal 41 Victoria Parade, Fitzroy structure of the complex between the inhibitor and ribonuclease A. The structure was determined by molecular replacement and refined to an R cryst Victoria 3065, Australia of 19.4% at 2.5 Å resolution. Ribonuclease A binds to the concave region 2 Howard Hughes Medical of the inhibitor protein comprising its parallel -sheet and loops. The Institute and Department of inhibitor covers the ribonuclease active site and directly contacts several Biochemistry, University of active-site residues. The inhibitor only partially mimics the RNase- Texas Southwestern Medical nucleotide interaction and does not utilize the p1 phosphate-binding Center, 5323 Harry Hines pocket of ribonuclease A, where a sulfate ion remains bound. The 2550 Å 2 Blvd., Dallas, TX 75235-9050 of accessible surface area buried upon complex formation may be one of USA the major contributors to the extremely tight association (K i = 5.9 × 10 -14 M). The interaction is predominantly electrostatic; there is a high chemical complementarity with 18 putative hydrogen bonds and salt links, but the shape complementarity is lower than in most other protein–protein complexes. Ribonuclease inhibitor changes its conformation upon complex formation; the conformational change is unusual in that it is a plastic reorganization of the entire structure without any obvious hinge and reflects the conformational flexibility of the structure of the inhibitor. There is a good agreement between the crystal structure and other biochemical studies of the interaction. The structure suggests that the conformational flexibility of RI and an unusually large contact area that compensates for a lower degree of complementarity may be the principal reasons for the ability of RI to potently inhibit diverse ribonucleases. However, the inhibition is lost with amphibian ribonucleases that have substituted most residues corresponding to inhibitor-binding residues in RNase A, and with bovine seminal ribonuclease that prevents inhibitor binding by forming a dimer.  1996 Academic Press Limited Keywords: ribonuclease inhibitor; RNase A; leucine-rich repeats; crystal structure; interaction *Corresponding author Introduction The formation of specific protein-protein com- plexes is of fundamental importance in most cellular processes. Certain structural motifs, for example the immunoglobulin fold (Williams & Barclay, 1988), the epidermal growth factor-like module (Campbell & Bork, 1993) and the kringle domain (Furie & Furie, 1988), are widely used in diverse molecular recognition processes and appear to have particularly useful characteristics for involvement in protein-protein interactions. These and many other such motifs have been recruited as modules in mosaic proteins by exon shuffling and duplication (Patthy, 1991). The recently characterized leucine-rich repeat (LRR) is likely to be another structural motif widely used in molecular recognition. LRRs have been found in over 60 different proteins with diverse functions and cellular locations that all appear to be Abbreviations used: LRR, leucine-rich repeat; RI, ribonuclease inhibitor; RNase, ribonuclease; 3-D, three-dimensional; BS-RNase, bovine seminal RNase; DTT, dithiothreitol; Ki, inhibition constant; Hepes, N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]; EDN, eosinophil-derived neurotoxin; PC, Patterson correlation. 0022–2836/96/501028–16 $25.00/0  1996 Academic Press Limited