Crystal structure of human p120 homologue protein PH1374 from Pyrococcus horikoshii

Introduction. Human p120 is a nucleolar proliferationassociated antigen, and has been detected in the nucleoli of a variety of malignant tumors, including cancers of the breast, liver, gastrointestinal tract, genitourinary tract, blood, lymph system, lung, and brain. It contains 855 amino acid residues, and its central hydrophobic domain (residues 380 –583) is conserved among many organisms. Expression of p120 is increased 60to 80-fold in several tumor cells as compared to normal resting cells and correlates with the tumor growth rate. Furthermore, p120 is expressed in the G1 phase of the cell cycle and its level peaks during S phase. In 1994, using computer methods for identification of amino acid motifs in sequence databases and multiple alignment, it was suggested that the central domain (380 –583) of p120 and its homologues comprises a new family of S-adenosylL-methyonine (SAM)-utilizing methyltransferases (MTase). A sequence homologue of the p120 central domain from Saccharomyces cerevisiae, Nop2p, was thought to play a direct role in pre-25S rRNA processing and 60S subunit production and yeast viability. Another homologous protein from Escherichia coli, Fmu, was reported to catalyze transference of a methyl group to carbon 5 of a pyrimidine nucleotide at position 967 cytosine of E. coli 16S rRNA; this represents the first RNA mC MTase whose amino acid sequence is known. In this context, this series of p120 homologue proteins is thought of as an RNA mC MTase family. PH1374 from Pyrococcus horikoshii OT3 consists of 315 amino acids. This protein has 16% identity to all amino acids of human p120, but near the central domain of p120, it has 41% identity in 288 aa overlap (p120 residues 300–581, PH1374 residues 35–315). Though it is known that four genes are paralogues to PH1374 in P. horikoshii genomes by COGs database search, of the five proteins, PH1374 has the highest similarity to p120. Also, proteins that have sequence homology with human p120 characteristically contain two highly conserved cysteines. These cysteines are thought of as essential to the protein’s function, but studies with Nop2p and Fmu seemed to be contradictory in terms of which residue acts as the activesite nucleophile in covalent catalysis. Materials and Methods. The PH1374 gene was amplified by polymerase chain reaction (PCR) from the genomic DNA of P. horikoshii, and cloned into a pET-22b( ) vector (Novagen). The (His)6-tagged recombinant PH1374 was expressed in cells of the E. coli strain BL21-StarTM(DE3)RIL (Stratagene), and two additional amino acids (leucine and glutamic acid) derived from the vector were inserted between the last amino acid of the protein and the (His)6-tag. The cells were grown at 37°C in 6 L Luria– Bertani (LB) medium containing 50 g/mL ampicillin and 34 g/mL chloramphenicol. Expression of recombinant protein was induced by adding 0.4 mM isopropyl-Dgalactoside (IPTG). After IPTG injection, E. coli was cultured for 15 h with shaking. Cells were harvested, then disrupted using a French press in A buffer (50 mM sodium phosphate pH 8.0, 300 mM NaCl). Cell debris was removed by centrifugation and the supernatant was mixed to Ni-NTA resin (Qiagen) and eluted using a linear gradient of 0.0–0.4 M imidazole. The fractions containing PH1374 were dialyzed against buffer B (10 mM sodium phosphate pH 8.0, 200 mM NaCl) and applied to a DE52 (Whatman) equilibrated with buffer B. PH1374 was passed through DE52, and contaminants were adsorbed. The purified protein was concentrated to 5 mg/mL. The selenomethionine (Se-Met) derivative was also expressed in BL21StarTM(DE3)-RIL and purified under almost the same conditions as the native protein. The crystallization conditions for PH1374 consisted of 0.1 M citric acid pH5, 15% PEG6K, 1% 1-5 pentandiol, and 5% glycerol. Crystals were grown at 20°C to a size of up to 0.3 mm 0.04 mm 0.01 mm within a few months. X-ray diffraction data of Se-Met PH1374 were collected to 1.9 Å resolution at 100 K at the BL41XU in SPring-8 (Hyogo,