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We describe the mechanism of ribonuclease inhibition by ribonuclease inhibitor,
a protein built of leucine-rich repeats, based on the crystal structure of the
complex between the inhibitor and ribonuclease A. The structure was determined
by molecular replacement and refined to an Rcryst of 19.4% at 2.5 A resolution.
Ribonuclease A binds to the concave region of the inhibitor protein comprising
its parallel beta-sheet and loops. The inhibitor covers the ribonuclease active
site and directly contacts several active-site residues. The inhibitor only
partially mimics the RNase-nucleotide interaction and does not utilize the p1
phosphate-binding pocket of ribonuclease A, where a sulfate ion remains bound.
The 2550 A2 of accessible surface area buried upon complex formation may be one
of the major contributors to the extremely tight association (Ki = 5.9 x 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.
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