Selective abolition of pancreatic RNase binding to its inhibitor protein

Abstract

We have modified RNase inhibitor (RI) protein so that it no longer detectably binds pancreatic RNases but retains near-native affinity for human angiogenin (ANG). The K(i) value for RNase A is increased by a factor of >10(8), from 36 fM to >4 microM, and the selectivity factor for ANG is now >10(9). This dramatic change was achieved by remodeling the human RI loop segment Cys-408 -Leu-409 -Gly-410, which makes minor interactions with pancreatic RNase but does not contact ANG. The modifications selected were designed to sterically hinder docking of the undesired ligand. Three of the variants tested (C408W, G410W, and C408W/G410W) bind RNase A with almost the same avidity as WT RI. However, combination of the 408/410 double Trp replacement with deletion of the intervening residue, Leu-409, was sufficient to abolish inhibition of RNase A and human pancreatic RNase. The K(i) value for ANG with the deletion variant is 1.1 fM, only 2-fold higher than with WT RI. This variant may have potential utility both as an anticancer drug targeting ANG and as a tool for the investigation of the biological function of ANG. More generally, these findings demonstrate that a protein-protein interaction can be effectively and specifically disrupted by redesigning an interface region that makes no major energetic contribution to complex stability. This finding, in turn, may have implications for the development of small molecules that modulate protein-protein interactions.

Fig. 1.

(A) The crystal structure of pRI in its complex with RNase A (16), showing the α-carbon trace, the side chains of the 28 residues that contact RNase A, and all main-chain atoms of contact residues 408-411. Hot spot residues whose replacement decreased the binding free energy by >2.5 kcal/mol (20) are shown in red, the 408-411 segment is in blue, and other contact residues are in green. (B) Region of the pRI-RNase A crystal structure containing the 408-411 segment of pRI (standard colors) and residues 66-69 of RNase A (red), with hydrogen bonds indicated by broken lines. ANG residues 66-69 (blue) from the hRI-ANG complex (17) are superimposed.

Fig. 2.

Effects of (His)₆-pep-C408W/ΔL409/G410W hRI preparation from Ni-nitrilotriacetic acid column on the enzymatic activities of T80A/Q117A-ANG (▴) and RNase A (•). The volumes of eluate indicated were taken from a total of 1.2 ml that contained the majority of the variant from 1 liter of bacterial culture.

Fig. 3.

Dissociation of the complexes of WT and C408W/ΔL409/G410W hRI fusion proteins with ANG (• and ▴, respectively) and EDN (▾ and ▪, respectively). Data were normalized with respect to the amount of dissociation at the first time point (3-10% of the total). Lines are fits of the data to a single-exponential decay.

Fig. 4.

Model of C408W/ΔV409/G410W pRI (green) superimposed onto the crystal structure of RNase A (purple) in the pRI-RNase A complex (16).