PH0828 is a hypothetical protein from a hyperthermophilic archaebacterium, Pyrococcus horikoshii OT3. A BLAST1 database search revealed that homologues of PH0828 are conserved among many organisms, primarily archaea and bacteria, and some eukarya also have proteins homologous with PH0828; such conservation suggests that PH0828 and its homologues play some essential role in these organisms. To date, none of the PH0828 homologues have been assigned a function. BtrG, a homologue of PH0828 from Bacillus circulans, still remains uncharacterized, but its participation in a pathway in this organism has been implicated. The gene encoding BtrG is part of a butirosin-biosynthetic gene cluster, which consists of at least 17 genes.2, 3 The functions of most genes in this cluster are not well understood. PH0828 shares approximately 28% identity with all amino acids of BrtG. To reveal the functions of PH0828 and its homologues, we determined the three-dimensional (3D) structure of PH0828 at 1.4 Å resolution.
Materials and Methods.
The PH0828 gene was amplified by polymerase chain reaction (PCR) from the genomic DNA of P. horikoshii OT3. The PCR amplicon was digested with NdeI/XhoI and the digested product was cloned into the corresponding site of the pET-26b(+) vector (Novagen Madison, WI), leading to a C-terminal His-tagged protein. The His-tag consisted of 8 residues, and the sequence was LEHHHHHH. Recombinant proteins were expressed in E. coli strain BL21-Codon Plus(DE3)-RIL (Stratagene, La Jolla, CA). Cells were grown at 37°C in 6 L LB medium containing 50 μg ml−1 kanamycin and 34 μg ml−1 chloramphenicol. The expression of PH0828 was induced by 1 mM isopropyl-β-D-thiogalactoside (IPTG). After IPTG injection, the medium was incubated at 37°C for 5 h with shaking. The cells were harvested (4,000g, 15 min at 4°C) and disrupted using a French press at 1,200 psi in buffer A (50 mM sodium phosphate, pH 8.0, 300 mM NaCl). The cell debris was removed by centrifugation (40,000g, 30 min at 4°C). The supernatant was incubated at 70°C for 30 min and then centrifuged (40,000g, 30 min at 4°C). Three mL of Ni-NTA resin (Qiagen, Chatsworth, CA) equilibrated with buffer A was added to the supernatant and the sample was mixed for 1 h at 4°C. Then, Ni-NTA resin was packed in a syringe column. The column was washed with buffer B (50 mM sodium phosphate, pH 6.0, 300 mM NaCl, 10 mM imidazole) and PH0828 was eluted with buffer C (50 mM sodium phosphate, pH 6.0, 300 mM NaCl, 250 mM imidazole). All fractions containing PH0828 were pooled and dialyzed against buffer A and then applied to a HiLoad 26/60 Superdex 75pg column (Amersham Bioscience, Arlington Heights, IL) equilibrated with buffer A. PH0828 was eluted as a single peak, and the fractions containing PH0828 were pooled and dialyzed against 10 mM Tris-HCl, pH 9.0. Then, PH0828 was concentrated to 21 mg/ml by means of a centrifugal concentrator.
The crystallization conditions for PH0828 consisted of 0.1 M CHES, pH 9.5, and 1.0 M sodium citrate, and crystals were grown to a size of 0.2 × 0.2 × 0.2 mm at 20°C within a few days. Au derivative crystals of PH0828 were prepared by soaking the crystals in 10 mM KAu(CN)2 for 6 days at 20°C. X-ray diffraction data sets of native and the Au derivative were collected at the BL41XU beamline at SPring-8 (Hyogo, Japan) and beamline NW12 at the Photon Factory-Advanced Ring (Tsukuba, Japan), respectively. All measurements were carried out under cryogenic conditions (100 K) after the crystals were soaked in cryo-protectant solution with 32% (w/v) sucrose. Multiple-wavelength anomalous diffraction (MAD) data were collected using three different wavelengths (1.0391, 1.0401, and 1.2000 Å) from a single crystal of the Au derivative. The crystals of native and the Au derivative were found to belong to the space group P3121 and an asymmetric unit contained one PH0828 molecule. The cell dimensions of the native crystal were a = b = 51.7 Å, c = 81.5 Å, and γ = 120°, and those of the Au derivative were a = b = 51.0 Å, c = 79.8 Å, and γ = 120°. The data for the native crystal were processed using MOSFLM4 and SCALA,5 and those for the Au derivative were HKL2000.6
One gold atom positioned at the crystallographic 2-fold rotational axis was determined using SOLVE.7 The heavy-atom parameters were refined and phase calculations were carried out using SHARP.8 The initial phases were improved by solvent flattening with SOLOMON9 implemented in SHARP. Based on the initial electron density map, 102 of 124 residues were built using the graphic program O.10 After the first round of refinement with the program CNS11 using the MAD data for the Au derivative, the model was further refined using native data to achieve a resolution of up to 1.4 Å. The summary of the data statistics is presented in Table I.
Table I. Summary of Data Collection and Refinement Statistics†
Values in parentheses are for the outermost resolution shell.Rmeas = Σh[m/(m − 1)]1/2Σj|〈I〉h − Ih,j|/ΣhΣjIh,j, here 〈I〉h is the mean intensity of symmetry-equivalent reflections and m is redundancy. Rmerge = ΣhΣj|〈I〉h − Ih,j|/ΣhΣjIh,j, where 〈I〉h is the mean intensity of symmetry-equivalent reflections.
Wave length (Å)
a = b = 51.7 Å, c = 81.5 Å, and γ = 120°
a = b = 51.0 Å, c = 79.8 Å, and γ = 120°
No. of non-hydrogen atoms
RMSD bond length (Å)
RMSD bond angle (deg.)
In most favored regions (%)
In additional allowed regions (%)
Results and Discussion.
The final model included 110 of 116 residues of PH0828, 8 residues of the His-tag, and 181 water molecules [Protein Data Bank (PDB) code: 1V30]. The atomic models of the six N-terminal residues could not be built due to poor electron density. The 3D structure of PH0828 revealed an α+β-fold consisting of four α-helices and seven β-strands [Fig. 1(A)]. The secondary structure elements were ordered as follows: β1-α1-β2-β3-β4-β5-α2-α3-β6-β7-α4. Five β-strands (β1, β2, β5, β6, β7) formed a central β-sheet, and three α-helices (α1, α2, α3) and one β-sheet (β3, β4) located surrounding the central β-sheet. A C-terminal long α-helix (α4) constructed by Phe112-Arg116 of PH0828 and the His-tag (LEHHHHHH) protruded outside the molecule. The helix α4 created the conditions for van der Waals interactions at the monomer-monomer interface of the dimer structures formed by the crystallographic 2-fold rotational axis. This interaction may be important for growing crystals; we were unable to obtain any crystals of wild-type PH0828 under the same crystallization conditions. A search using the DALI12 server did not show any structures that were significantly similar to the 3D structure of PH0828.
The present analysis also revealed that the PH0828 molecule had a cavity surrounded by its central β-sheet and two α-helices (α1 and α3). The length of this cavity was approximately 10 Å, and the width was about 8 Å. From the Fo-Fc map, it was determined that 2-(n-cyclohexylamino)ethane sulfonic acid (CHES), which was included in the crystallization buffer, was present in this cavity [Fig. 1(B)]. This cavity was found to have two entrances, one of which was constructed by main-chains from Asp73 to Leu83 and a side chain of Trp96, and another entrance constructed by the side-chains of Thr15, Leu22, Leu47, Ile75, and Ile77. Both entrances were large enough for the passage of the six-member ring of the CHES molecule. The results of the multiple sequence alignment of PH0828 and its sequence homologues (Fig. 2) revealed that most residues located at this cavity were highly conserved. For example, Tyr13, Gly14, and Leu16 were completely conserved, and Thr15, Tyr81, and Trp96 had conserved properties in their side chains among these homologues. Such conservation suggests that this cavity plays an important role in PH0828 activity. BtrG, a homologous protein of PH0828 from Bacillus circulans, is thought to participate in the butirosin biosynthetic pathway starting with D-glucose. Most of the products in this pathway contain a six-member ring, and the CHES molecule also contains a six-member ring. A description of the CHES binding mechanism of PH0828 may aid in the search for the intrinsic substrate of PH0828 and its homologous protein, BtrG.
We thank M. Kawamoto and H. Sakai for their kind assistance with the data collection at beamline BL41XU of the SPring-8 facility. We are also grateful to M. Suzuki, N. Igarashi, and N. Matsugaki for their kind help with the data collection at beamline NW12 of the Photon Factory-Advanced Ring. This work was supported by a research grant from the National Project on Protein Structural and Functional Analyses from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.