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X-Ray Crystallographic Structure of Rnase Po1 That Exhibits Anti-Tumor

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X-Ray Crystallographic Structure of Rnase Po1 That Exhibits Anti-Tumor 968 Regular Article Biol. Pharm. Bull. 37(6) 968–978 (2014) Vol. 37, No. 6 X-Ray Crystallographic Structure of RNase Po1 That Exhibits Anti- tumor Activity Hiroko Kobayashi,*,a Takuya Katsutani,b Yumiko Hara,b Naomi Motoyoshi,a Tadashi Itagaki,a Fusamichi Akita,b Akifumi Higashiura,b Yusuke Yamada,c Norio Inokuchi,a and Mamoru Suzuki*,b a School of Pharmacy, Nihon University; 7–7–1 Narashinodai, Funabashi, Chiba 274–8555, Japan: b Institute for Protein Research, Osaka University; 3–2 Yamadaoka, Suita, Osaka 565–0871, Japan: and c Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization KEK; 1–1 Oho, Tsukuba, Ibaraki 305–0801, Japan. Received November 29, 2013; accepted March 12, 2014 RNase Po1 is a guanylic acid-specific ribonuclease member of the RNase T1 family from Pleurotus os- treatus. We previously reported that RNase Po1 inhibits the proliferation of human tumor cells, yet RNase T1 and other T1 family RNases are non-toxic. We determined the three-dimensional X-ray structure of RNase Po1 and compared it with that of RNase T1. The catalytic sites are conserved. However, there are three disul- fide bonds, one more than in RNase T1. One of the additional disulfide bond is in the catalytic and binding site of RNase Po1, and makes RNase Po1 more stable than RNase T1. A comparison of the electrostatic po- tential of the molecular surfaces of these two proteins shows that RNase T1 is anionic whereas RNase Po1 is cationic, so RNase Po1 might bind to the plasma membrane electrostatically. We suggest that the structural stability and cationic character of RNase Po1 are critical to the anti-cancer properties of the protein. Key words ribonuclease; crystal structure; anti-tumor activity; Pleurotus ostreatus RNase Po1 hydrolyzes single-stranded RNA via a 2′,3′-cy- not RNase Po1 has three disulfide bonds, and which cysteine clic phosphate intermediate at the 3′-terminus of oligonucle- residues may form those bonds. Furthermore, RNase Po1 is otides, and is a guanylic acid-specific ribonuclease (RNase) (a an alkaline protein (isoelectric point (pI) 9.0), whereas RNase member of the RNase T1 family of RNases). RNase Po1 has a T1 (pI 2.9) and most members of the RNase T1 family of molecular mass of approximately 11 kDa and exhibits optimal RNases are acidic proteins (pI 4.0–4.5).24,25) Thus, RNase Po1 activity at pH 7.5, similar to RNase T1 from Aspergillus ory- is a unique member of the RNase T1 family. Recently, we zae, the best-known member of this family.1,2) We previously reported that RNase Po1 exhibits anti-tumor activity towards isolated and purified RNase Po1 from Pleurotus ostreatus several types of human tumor cells,26) in contrast to RNase of the basidiomycota and reported its amino acid sequence.1) T1 and other RNase T1 family RNases that are non-toxic to There is high sequence identity (40%) between RNase Po1 and tumor cells, except for α-sarcin from Aspergillus giganteus.27) RNase T1. The X-ray crystallographic structures of RNase α-Sarcin is a single polypeptide chain protein composed of T13) and RNase Ms4) have been reported, and the catalytic site 150 amino acids, bigger than RNase Po1 (101 amino acids) and base recognition region for RNase activity have been elu- and with low sequence identity.28) α-Sarcin exhibits anti-tumor cidated. The catalytic site of RNase T1 consists of His40 and/ activity by degrading the larger ribosomal RNA of tumor or Glu58, Arg77, and His92.5) Moreover, Steyaert et al.6) and cells.29) The three-dimensional structure of α-sarcin has been Nonaka et al.7) reported that Glu58, rather than His40, must solved,30) and it is necessary to compare its structure with be the general base catalyst of the RNase T1 family. The base that of RNase Po1 to understand the basis of their anti-tumor recognition site of RNase T1 consists of Tyr42, Asn43, Asn44, activities. Tyr45, Glu46, and Asn98.5) These residues are completely There is another RNase family whose members have conserved in RNase Po1 except for Tyr45 (RNase T1) being molecular masses of 13–14 kDa. This family is pyrimidine changed to Phe (RNase Po1). A comparison of the primary base-specific and is referred to as the RNase A family. Al- structures of RNase Po1 and RNase T1 and other RNase T1 most all RNase A family members are non-toxic, but some family RNases shows that RNase T1 contains four cysteine RNases have been reported to exhibit anti-tumor activity.31–35) residues that form two disulfide bonds, whereas RNase Po1 RNases from Rana pipiens are the most extensively studied, has six cysteine residues1,8–23) (Fig. 1). The C9–C99 disulfide and ranpirnase (Onconase) is currently in clinical trials as an bond of RNase Po1 is superimposable on the analogous di- anti-tumor drug.36,37) The X-ray crystallographic structures of sulfide bond in RNase T1 (C6–C103). This disulfide bond is RNase A and Onconase have been reported.38,39) Comparisons conserved in all known RNase T1 family enzymes, except for of their structures suggest a relationship between stability and those of bacterial origin. The C48–C82 bond of RNase Po1 anti-tumor activity.40,41) In the present work, we determined is superimposable on RNase U1 and RNase U2 from Usti- the X-ray crystallographic structure of RNase Po1 and in- lago sphaerogena. Therefore, these two disulfide bonds may vestigated the relationship between structure and anti-tumor exist in RNase Po1. The other cysteine residues in RNase Po1 activity by comparing the X-ray structures of RNase Po1 and (Cys7, Cys84) are not found in RNase T1 and other RNase T1 RNase T1, which have high sequence identity. family RNases. Therefore, we have not considered whether or The authors declare no conflict of interest. * To whom correspondence should be addressed. e-mail: [email protected]; © 2014 The Pharmaceutical Society of Japan [email protected] June 2014 969 Fig. 1. Comparison of the Amino Acid Sequences of RNase Po1 or RNase T1 with Those of RNases Belonging to the RNase T1 Family Po1: Pleurotus ostreatous RNase,1) U1, U2: Ustilago sphaerogena RNase,8,9) F1: Fusarium moniliforme RNase,10) FL1: Fusarium lateritium RNase,11) Th1: Trichoderma harzuanum RNase,12) Ms: Aspergillus saitoi RNase,13) T1: Aspergillus oryzae RNase,14) C2: Aspergillus clavatus RNase,15) Ap1: Aspergillus pallidus RNase,16) N1: Neu- rospora crassa RNase,17) Pch1: Penicillium crysogenum RNase,18) Pb1: Penicillium brevicompactum RNase,19) Sa: Streptomyces aureofaciens RNase,20) St: Streptomyces rythreus RNase,21) Bi: Bacillus intermedius RNase,22) Ba: Bacillis amyloliquefacience RNase.23) * Catalytic site. # Base recognition site. Disulfide bonds of RNases are shown as connected by bold lines. MATERIALS AND METHODS Table 1. Data Collection and Refinement Statistics Enzymes RNase Po1 was expressed in Escherichia coli. Parameter RNase Po1 The cDNA was ligated to expression vector pET-pel-Po1, Data collection constructed following the procedure of Huang et al.42) from Beamline Photon Factory BL-17A pET22b (Novagene, Darmstadt, Germany), then transferred to Wavelength (Å) 0.9800 E. coli BL21(DE3) pLysS (Novagene). The cells were cultured Space group P31 in Terrific Broth at 25°C for 7 d, with the addition of 100 µg/ Cell dimension (Å) a=b=75.56, c=34.80 mL of ampicillin and a final concentration of 0.5 mM isopropyl Resolution (Å) 37.78–1.85 (1.95–1.85) β-D-1-thiogalactopyranoside (IPTG) (Wako Pure Chemical Reflections measured 136098 Industries, Ltd., Osaka, Japan). The supernatant of the culture Unique reflections 18553 was used for subsequent purification steps. The supernatant Redundancy 7.7 (5.3) was fractionated with 90% saturated ammonium sulfate, and Completeness (%) 99.6 (84.7) a) the precipitate was collected by centrifugation at 10000 rpm Rmerge (%) 15.7 (62.7) Mean I/σ(I) 10.1 (2.5) for 30 min. The precipitate was suspended in 10 mM acetate buffer (pH 6.0) and dialyzed overnight against de-ionized Refinement Resolution (Å) 32.72–1.85 water. The dialysate was heated at 60°C for 10 min and then Number of reflections 18541 rapidly cooled in ice-cold water for 10 min. The precipitate R b)/R c) 0.164/0.176 was recovered by centrifugation at 10000 rpm for 30 min. Sub- work free R.m.s deviations sequent purification steps were carried out using previously 26) Bond length (Å) 0.0034 described protocols. Bond angles (°) 0.782 Enzyme Assay RNase activity was measured as de- Ramachandran analysis scribed previously using yeast RNA (Marine Biochemicals, Favored/allowed (%) 98.7/1.3 Tokyo, Japan) as the substrate at pH 7.5 at 37°C.43) Values in parentheses are for the highest-resolution shell. a) Rmerge=∑hkl ∑i |I(hkl; i)− Protein Concentration The protein concentration of the 〈I(hkl)〉|/∑hkl ∑i I(hkl; i), where I(hkl; i) is the intensity of an individual mea- final enzyme preparation was determined spectrophotometri- surement, and 〈I(hkl)〉 is the average intensity from multiple observations. b) R =S||F |−|F ||/|F |, where |F | and |F | are the observed and calculated cally, assuming an absorbance of 0.54 for a 0.1% solution at work obs calc obs obs calc structure factor amplitudes, respectively. c) Rfree is the same as Rwork, but for a 10.0% 280 nm. This value was estimated from the amino acid com- subset of all reflections for RNase Po1. position of RNase Po1 (data not shown). Tricine-Sodium Dodecyl Sulfate-Polyacrylamide Gel (3.5 M sodium formate, 0.1 M 1,3-bis[tris(hydroxymethyl)- Electrophoresis (Tricine-SDS-PAGE) Tricine-SDS-PAGE methylamino] propane-1,3-diol (Bis-Tris propane) (pH 7.0) in was performed using a 15% polyacrylamide gel by Schagger’s SaltRx1 (Hampton Research, Aliso Viejo, CA, U.S.A.).
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