Structural analysis of the transcriptional regulator homolog protein from Pyrococcus horikoshii OT3.

Structural analysis of the transcriptional regulator homolog protein from Pyrococcus horikoshii OT3 Introduction PH1061 is a hypothetical protein of 100 residues (11.4 kDa) from the hyperthermophilic archaebacterium, Pyrococcus horikoshii OT3 1 , and is conserved among many archaeal and bacterial organisms. Although the functions in these organisms are still unknown, a Pfam 2 database search revealed that PH1061 has a helix-turn-helix (HTH) motif (" HTH_5 motif " in the Pfam database), and suggested that it is a DNA-binding protein, like members of the ArsR family 3. ArsR is a transcriptional repressor of the arsenic resistance operon in Escherichia coli and other members of this family involve zinc-sensing transcriptional repressors, such as CzrA 4 from Staphylococcus aureus and SmtB 5 from Synechococcus pcc7942. The primary sequence similarities to PH1061 are 20%, 21%, and 6.6% with ArsR, CzrA, and SmtB, respectively. There are 10 proteins with the HTH_5 motif in P. horikoshii, but none of their functions are known. For structure-based functional analysis, the three-dimensional structure of PH1061 was determined at a resolution of 2.05 Å using the multi-wavelength anomalous diffraction (MAD) method. Materials and Methods The PH1061 gene was amplified by polymerase chain reaction (PCR) from P. horikoshii OT3 genomic DNA. The PCR product was cloned into the pET-22b (+) vector (Novagen). PH1061 protein was expressed in E. coli strain B834 (DE3) as a C-terminal histidine-tagged fusion protein. Cells were grown in Luria-Bertani (LB) medium at 37°C and induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The cells were harvested by centrifugation, washed with buffer A (50 mM sodium phosphate pH 6.0, 300 mM NaCl), and disrupted using a French press. Cell debris was removed by centrifugation, and the supernatant was incubated at 70°C for 30 min, then denatured proteins from host E. coli cells were removed by centrifugation. The extract was mixed to with Ni-nitrilo triacetic acid (Ni-NTA) resin (QIAGEN), washed with buffer A containing 20 mM imidazole, and the bound proteins were eluted by adding 500 mM imidazole. The eluate was dialyzed against buffer A, and loaded onto a HiLoad 26/60 Superdex 75pg column (Amersham Bioscience) equilibrated with buffer A for size exclusion chromatography. Fractions containing PH1061 were pooled and applied to a POROS QE/M column (Applied Biosystems) equilibrated with buffer A. PH1061 passed through the column and contaminants were adsorbed. The flow-through fractions were pooled, buffer A was exchanged for buffer B (20 mM 2-(N-morpholino) ethanesulfonic acid (MES) pH 6.0, …