X-ray Structure of Two Crystalline Forms of aStreptomycete Ribonuclease with Cytotoxic Activity

Jozef Sevcik,Lubica Urbanikova,Peter A Leland,Ronald T Raines

doi:10.1074/jbc.m208425200

Abstract

Ribonuclease (RNase) Sa3 is secreted by the Gram-positive bacterium Streptomyces aureofaciens. The enzyme catalyzes the cleavage of RNA on the 3' side of guanosine residues. Here, x-ray diffraction analysis was used to determine the three-dimensional structure of two distinct crystalline forms of RNase Sa3 to a resolution of 2.0 and 1.7 A. These two structures are similar to each other as well as to that of a homolog, RNase Sa. All of the key active-site residues of RNase Sa (Asn(42), Glu(44), Glu(57), Arg(72), and His(88)) are located in the putative active site of RNase Sa3. Also herein, RNase Sa3 is shown to be toxic to human erythroleukemia cells in culture. Like onconase, which is an amphibian ribonuclease in Phase III clinical trials as a cancer chemotherapeutic, RNase Sa3 is not inhibited by the cytosolic ribonuclease inhibitor protein. Thus, a prokaryotic ribonuclease can be toxic to mammalian cells.

Highlights

Like onconase, which is an amphibian ribonuclease in Phase III clinical trials as a cancer chemotherapeutic, RNase Sa3 is not inhibited by the cytosolic ribonuclease inhibitor protein
RNase Sa was the first ribonuclease to be isolated from the growth medium of a Gram-positive microorganism, Streptomyces aureofaciens [1]
The ability of secretory ribonucleases from human [21], cow [22,23,24], bull [25,26,27], and frog [28] to evade ribonuclease inhibitor (RI) endows them with toxicity for mammalian cells

Summary

EXPERIMENTAL PROCEDURES

Protein Crystallization—Protein production, purification, and crystallization were performed as described previously [29]. Trigonal crystals (crystal I) in the P3121 space group with unit-cell dimensions of a ϭ b ϭ 64.72 Å, c ϭ 69.57 Å, and ␤ ϭ 120° were grown using 0.10 M Hepes buffer, pH 7.6, containing Li2SO4 (1.6 M) as the precipitant solution. Tetragonal crystals (crystal II) in the P41212 space group with unit-cell dimensions of a ϭ b ϭ 34.05 Å and c ϭ 147.22 Å were grown using 2-methyl-2,4-pentanediol (10%, v/v) and ammonium sulfate (40 mM) as the precipitant solution. Data from crystal I were collected to 2.0 Å at 100 K using synchrotron facilities at EMBL (Hamburg, Germany), and data from crys-.

Side chain

Structure II

RESULTS AND DISCUSSION