The full-length hisG and hisZ genes were cloned from T. maritima genomic DNA using the following polymerase chain reaction (PCR) primers: 5′-GGGAAACATATGCTGA AACTGG-3′ (hisG 5′ primer); 5′-sGAGCAAGCTTTTATCC CCGG-3′ (hisG 3′ primer); 5′-CTGGGGCCATGGATTTCT TGG-3′ (hisZ 5′ primer); and 5′-CCTTTGGGAAT TCCCAGTTTCAG-3′ (hisZ 3′ primer). Expression plasmids were constructed by digesting the hisG PCR fragment (643 bp) with NdeI and HindIII and inserting the product into pET21, which harbours an ampicillin resistance cassette. Likewise, the hisZ PCR fragment (841 bp) was digested with NcoI and EcoRI and ligated into pET28, which contains a kanamycin resistance cassette.
Protein expression and purification
For expression of the native genes, E. coli BL21(DE3) RIL cells were transformed with the hisGS or hisZ expression constructs, and plated out on Luria–Bertani (LB) plates containing 50 µg ml−1 ampicillin or 25 µg ml−1 kanamycin, respectively, at 37°C. Before the final volume expression experiments, 25–50 ml of precultures were grown from a single colony overnight in LB at 37°C. A 10 ml aliquot of this preculture was used as an inoculum to 1 l of expression culture in a 5 l flask. For native expression, LB medium supplied with the appropriate antibiotics was used as growth medium. Isopropyl-β- d-thiogalactopyranoside (IPTG) (1 mM) was added to start induction at an OD600 of ≈0.6–0.7 and the cultures were harvested after another 4–6 h. The typical yield for tmHisG and tmHisZ was ≈15–25 mg of purified protein per litre of culture.
For production of the seleno- l-methionine (SeMet) incorporated protein, methionine auxotroph E. coli strain B834(DE3) cells were transformed with the hisGS and hisZ expression constructs, and plated out as described above. The following media and stock solutions were used: M9 minimal medium, 10× stock (0.55 M Na2HPO4, 0.30 M KH2PO4, 85 mM NaCl, 60 mM NH4Cl), medium A (1× M9 supplied with 20 mM d-(+)-glucose, 1 mM MgCl2, 0.3 mM CaCl2, 4 µM biotin, 2.7 µM thiamine), starvation medium (medium A plus antibiotics, without methionine), trace element solution, 100× stock (30 mM FeCl3·6H2O, 6.0 mM ZnCl2, 0.8 mM CuCl2·4H2O, 0.4 mM CoCl2·6H2O, 1.6 mM H3BO3, 0.07 mM MnCl2·6H2O), 50 mg ml−1l-methionine (1000× stock) and 50 mg ml−1 SeMet (1000× stock). A single colony of freshly transformed cells was used to inoculate a 25–50 ml preculture in medium A plus methionine, which was grown overnight at 37°C until saturation (OD600 ≈ 2–3). Next, the preculture was added to 1 l of medium A with methionine pre-incubated at 37°C and grown until OD600 of 1.0. The cell suspension was centrifuged twice at 4000 r.p.m. in a Sorvall SLA-3000 rotor and washed in 1× PBS. The cell pellet was then resuspended in 1 l of starvation medium and incubated at 37°C for 4–6 h. Thirty minutes before induction, 1 ml of SeMet was added. The cell culture was induced by the addition of 1 mM IPTG and expression proceeded for 6 h before harvesting. The typical yield for SeMet tmHisG and tmHisZ was ≈5–10 mg of purified protein per litre of culture.
Both the native and the SeMet proteins were purified in an identical way, with the exception of the addition of 1 or 5 mM DTT to all buffers respectively. The expression cultures were centrifuged at 4500 r.p.m. in a Sorvall SLA-3000 rotor, and the cell pellets washed with 1× PBS. In order to obtain a 1:1 protein complex, the wet cell pellets of tmHisGS and tmHisZ were mixed in a ≈1.5:1 (w/w) ratio. The combined cell pellets were then thoroughly resuspended in 25 ml of stabilizing buffer per litre of culture for heat shock treatment at 80°C for 10 min. The composition of the stabilizing buffer was as follows: 50 mM Tris-Cl, pH 8.0, containing 0.4 mM histidine, 4 mM K3PO4, 100 mM KCl, 5 mM DTT. The lysate was then clarified by centrifugation at 23 000 r.p.m. in a Sorvall SS-34 rotor and filtered through a 0.22 µm membrane. The binary tmHisGS–tmHisZ complex was further purified in 20 mM Tris/HCl, pH 7.5, by anion exchange chromatography on a Pharmacia HiLoad Q-Sepharose (HR 10/26), eluting in a NaCl linear gradient at about 300 mM NaCl. The elution fractions were pooled, concentrated and dialysed against a suitable buffer for gel filtration (20 mM Tris-Cl, pH 7.5, 40 mM NaCl, 0.4 mM histidine, 5 mM DTT, 0.6 mM AMP). Gel filtration was carried out on a Pharmacia Superdex 200 (HR 26/60) column, and the complex eluted with an estimated molecular mass of ≈200 kDa, corresponding to the (tmHisGS)4–(tmHisZ)4 octameric complex. The elution fractions matching the peak were pooled and concentrated up to 50 mg ml−1, and 0.6 mM AMP was added. Part of the purified ATP–PRTase complex was flash-frozen in liquid nitrogen and stored at −80°C. Protein samples for biochemical assays were produced by the same procedures but without histidine and AMP.
Steady-state kinetics were performed to determine the kinetic parameters of the tmATP–PRTase complex and the inhibition constant of the pathway product histidine. The steady-state assay was based on the increase of A290 after the formation of the product PR-ATP (Voll et al., 1967). Reactions contained 1.2 µM of enzyme (molarity calculated for a tmHisG–tmHisZ dimer) in 50 mM Tris (pH 8.0), 10 mM MgCl2, 150 mM KCl, inorganic pyrophosphatase at 2 µg ml−1, and saturating concentrations (5 mM) ATP, in a final volume of 120 µl. PRPP was added last (from 0.5 to 8.7 mM final concentration) to start the reaction, which was monitored over 10 min every 5 s in a UVIKON 922 spectrophotometer (Kontron) at 20°C. To measure the inhibitory effect of histidine, reactions were set up as described and histidine was added to a final concentration of 0.1, 1, 5 or 10 mM. These samples were incubated with histidine during 1 h before adding PRPP (Bell et al., 1974). The kcat and KmPRPPvalues were determined from saturation curves by non-linear least squares regression. KIHis was determined from the relation v0app = Vmapp[S]/(Kmapp+[S]), where Vmapp = k3[E0]/(1 + [I]/KI) and Kmapp = Km.
X-ray structure determination
Crystals of the binary complex were grown by assembling 2 µl of 8 mg ml−1tmATP–PRTase with 2 µl of reservoir solution containing 22.5% (w/v) methylpentanediol (MPD), 0.2 M phosphate/citrate buffer, pH 4.2, using the sitting drop vapour diffusion technique. Both the native and the SeMet crystals grew under identical crystallization conditions. Crystals suitable for diffraction appeared after 2 days and reached a final size of 1.0 × 0.45 × 0.35 mm. A stepwise increase of MPD concentration (up to 25% in 1% steps) was used to cryoprotect the crystals, which were flash-frozen on a liquid nitrogen stream.
Several X-ray data sets were collected on crystals of the native and SeMet tmATP–PRTase complexes (Table 1). A three-wavelength multiple anomalous dispersion data set was collected in two 0.2° steps and two 0.4° oscillation angle wedges per wavelength (over a total angular range of 158°) to avoid spot overlaps according to the data collection strategy program best (Popov and Bourenkov, 2003). The data were merged during data processing and scaled with denzo and scalepack (Otwinowski and Minor, 1997). We were able to identify 23 out of 28 selenium atoms with the software solve v2.03 (Terwilliger and Berendzen, 1999), using the single anomalous diffraction mode for the peak wavelength (0.9788 Å). The solution was refined against the inflection and remote data sets found. The final list of sites included the three out of the four possible selenium atoms at the N-terminus of each tmHisZ. The five missing sites corresponded to the fourth tmHisZ N-terminus (M1) and all four tmHisGS N-termini that are disordered. The figure of merit (fom) was 0.35 in the resolution range of 30–3.5 Å. Density improvement by density modification and non-crystallographic symmetry (NCS) averaging with resolve v2.03 substantially improved the quality of the electron density maps, and raised the fom to 0.52, using X-ray data from 30 to 2.6 Å. A correct, but incomplete, ATP–PRTase model was built automatically using the warpNtrace mode of ARP/wARP (Perrakis et al., 1999) with experimental phases up to 2.6 Å. The free R factor decreased from 43% to 40% and the work R factor from 38% to 25%, and the fom rose from 0.52 to 0.71, coupled with a significant increase in the quality of the electron density. The wARP model had a final (initial) connectivity index of 0.89 (0.79) and the total number of amino acids built was 1484 out of 1932 (77%), arranged in 27 fragments. The remaining model could be built manually, succeeded by one further round of ARP/wARP in molrep mode (Perrakis et al. 1999). The free R factor, work R factor and fom of the subsequent model were 34%, 22% and 0.76 respectively. The model was refined with cns v1.1 (Brunger et al. 1998) until convergence to a free R factor of 28.6% and a work R factor of 20.3% was achieved. The final model includes nearly all residues (1911 out of 1932). Its co-ordinates and the structure factors have been deposited in the Protein Data Bank data bank (1USY). Further statistics are summarized in Table 2.
Table 1. Crystallographic data and phasing statistics.
|Beamline||BW7A||BW6|| || |
|Space group||P212121||P212121|| || |
| a||100.7||101.6|| || |
| b||133.3||134.4|| || |
| c||152.5||154.2|| || |
|No. of reflections|
| Measured||818 378||930 472||930 829||1 133 033|
| Unique||62 204||52 743||52 833||65 941|
|Completeness (%)||86.9 (88.7)||98.4 (98.1)||98.2 (95.3)||95.3 (93.3)|
|〈I〉/σ(〈I〉)||13.8 (5.1)||18.2 (6.9)||18.4 (6.4)||16.0 (3.6)|
|R-sym (%)||3.9 (21.3)||5.5 (28.9)||5.4 (33.0)||6.0 (54.8)|
|R-anom (%)|| ||4.4 (15.1)||3.0 (16.6)||3.8 (27.3)|
|fom (mpe, °)|| ||0.34 (73.8)a||0.40 (73.8)b||0.52 (65.8)b|
Table 2. Refinement statistics of the tmATP–PRTase structure.
|No. of reflections||66 598|
|No. of reflections for validation||3547 (5.1%)|
|R free (%)||28.6|
|R work (%)||20.3|
|No. of non-H atoms|
| Protein||15 244|
|Bond length r.m.s.d. (Å)||0.016|
|Bond angle r.m.s.d. (°)||1.80|
|Mean B-value (Å2)|