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. 2007 Nov 13;46(45):13131-40.
doi: 10.1021/bi701521q. Epub 2007 Oct 23.

Intraspecies Regulation of Ribonucleolytic Activity

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Free PMC article

Intraspecies Regulation of Ribonucleolytic Activity

R Jeremy Johnson et al. Biochemistry. .
Free PMC article

Abstract

The evolutionary rate of proteins involved in obligate protein-protein interactions is slower and the degree of coevolution higher than that for nonobligate protein-protein interactions. The coevolution of the proteins involved in certain nonobligate interactions is, however, essential to cell survival. To gain insight into the coevolution of one such nonobligate protein pair, the cytosolic ribonuclease inhibitor (RI) proteins and secretory pancreatic-type ribonucleases from cow (Bos taurus) and human (Homo sapiens) were produced in Escherichia coli and purified, and their physicochemical properties were analyzed. The two intraspecies complexes were found to be extremely tight (bovine Kd = 0.69 fM; human Kd = 0.34 fM). Human RI binds to its cognate ribonuclease (RNase 1) with 100-fold greater affinity than to the bovine homologue (RNase A). In contrast, bovine RI binds to RNase 1 and RNase A with nearly equal affinity. This broader specificity is consistent with there being more pancreatic-type ribonucleases in cows (20) than humans (13). Human RI (32 cysteine residues) also has 4-fold less resistance to oxidation by hydrogen peroxide than does bovine RI (29 cysteine residues). This decreased oxidative stability of human RI, which is caused largely by Cys74, implies a larger role for human RI as an antioxidant. The conformational and oxidative stabilities of both RIs increase upon complex formation with ribonucleases. Thus, RI has evolved to maintain its inhibition of invading ribonucleases, even when confronted with extreme environmental stress. That role appears to take precedence over its role in mediating oxidative damage.

Figures

Figure 1
Figure 1
Structure and sequence of RI. (A) Ribbon diagram of the hRI (green)·RNase 1 (purple) complex (chains Y and Z from PDB accession code 1Z7X) (28). Images were made with the program PyMOL (DeLano Scientific, South San Francisco, CA). (B) Sequence alignment of the protein sequences of bRI and hRI. Conserved amino acids are shown with black boxes and contact residues (<3.9 Å) within the hRI·RNase 1 complex are shaded yellow (chains Y and Z from PDB accession code 1Z7X) (28). Alignments were performed with the program Clustal W (45). Consensus sequences for A-Type and B-Type repeats of RI-like LRRs were adapted from ref (26).
Figure 2
Figure 2
Dissociation of RI·ribonuclease complexes. The release of diethylfluorescein-labeled ribonuclease (100 nM) from RI (100 nM) was followed over time after the addition of a 50-fold molar excess of angiogenin (5 μM) (open symbols) or after the addition of PBS (closed symbols). Data points are the mean (±SE) of six separate measurements and are normalized for both the average fluorescence of rhodamine 110 (10 nM) and the fluorescence of unbound RNase A from hRI·RNase A. The initial fluorescence of unbound ribonuclease was used as the end-point for complete ribonuclease release (28). (A) Dissociation of bRI·RNase A (△) and hRI·RNase A (○); (B) Dissociation of bRI·RNase 1 (□) and hRI·RNase 1 (◇).
Figure 3
Figure 3
Conformation and conformational stability of RI·ribonuclease complexes. (A and C) Far-UV CD spectra of RI, ribonuclease, and RI·ribonuclease complexes (25 μM RI and 28 μM ribonuclease in PBS containing DTT (2 mM)). (B and D) Thermal denaturation of RI, ribonuclease, and RI·ribonuclease complexes (25 μM RI and 28 μM ribonuclease in PBS containing DTT (2 mM)). Molar ellipticity at 222 nm was monitored after a 2-min equilibration at each temperature. Data were fitted to a two-state model to determine values for Tm (Table 1) (42, 43). Panels A and B depict data for RNase A (△), bRI (□), bRI·RNase A (▲), and bRI·RNase 1 (■); panels C and D depict data for RNase 1 (◇), hRI (○), hRI·RNase 1 (◆), and hRI·RNase A (•).
Figure 4
Figure 4
Stability to hydrogen peroxide oxidation (H2O2) of RI and RI·ribonuclease complexes. The decrease in fluorescence intensity of diethylfluorescein-labeled ribonuclease upon binding to RI was used to measure the oxidative stability of free RI and RI·ribonuclease complexes (44, 36, 38). The complex formed by RI (10 μM) and ribonuclease (10 μM) was incubated in serial dilutions of H2O2 buffered in 20 mM HEPES–HCl buffer, pH 7.6, containing KCl (50 mM) and DTT (≤ 50 μM) for 30 min at 37 °C. Data points are the mean (± SE) of three independent experiments and were normalized for the maximum fluorescence of the unbound ribonuclease. Data were fitted to eq 2 to determine values of IC50 (Table 1). Panel A depicts data for bRI (□), bRI·RNase A (▲), and bRI·RNase 1 (■); panel B depicts data for hRI (○), hRI·RNase 1 (◆), and hRI·RNase A (•); panel C depicts data for hRI (○), C74L hRI (▼), and C408G hRI (▽).
Figure 5
Figure 5
Phylogenetic tree of RIs (A) and ribonucleases (B) from various organisms. Cladograms of RI and ribonuclease were made with the program MegAlign from the Lasergene software package (DNASTAR, Madison, WI). Bootstrap values were calculated using 2,000 replicates, and values >40 are reported. The bar indicates the distance for 0.10 substitutions per site. GenBank accession codes for RI and ribonuclease sequences are listed in the experimental procedures, and sequence alignments are shown in Figures S-2 and S-3.

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