ATP Analogues for Structural Investigations: Case Studies of a DnaB Helicase and an ABC Transporter

Nucleoside triphosphates (NTPs) are used as chemical energy source in a variety of cell systems. Structural snapshots along the NTP hydrolysis reaction coordinate are typically obtained by adding stable, nonhydrolyzable adenosine triphosphate (ATP) -analogues to the proteins, with the goal to arrest a state that mimics as closely as possible a physiologically relevant state, e.g., the pre-hydrolytic, transition and post-hydrolytic states. We here present the lessons learned on two distinct ATPases on the best use and unexpected pitfalls observed for different analogues. The proteins investigated are the bacterial DnaB helicase from Helicobacter pylori and the multidrug ATP binding cassette (ABC) transporter BmrA from Bacillus subtilis, both belonging to the same division of P-loop fold NTPases. We review the magnetic-resonance strategies which can be of use to probe the binding of the ATP-mimics, and present carbon-13, phosphorus-31, and vanadium-51 solid-state nuclear magnetic resonance (NMR) spectra of the proteins or the bound molecules to unravel conformational and dynamic changes upon binding of the ATP-mimics. Electron paramagnetic resonance (EPR), and in particular W-band electron-electron double resonance (ELDOR)-detected NMR, is of complementary use to assess binding of vanadate. We discuss which analogues best mimic the different hydrolysis states for the DnaB helicase and the ABC transporter BmrA. These might be relevant also to structural and functional studies of other NTPases.

Tris-HCl pH 8.0, 1 mM EDTA, and 300 mM sucrose. The protein-containing membranes were diluted at 2 g.L −1 and solubilized using 1% DDM (m/v) then centrifuged at 100,000 × g for 1 h. The supernatant was loaded onto a Ni-NTA column (Qiagen) previously equilibrated with 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 15% glycerol (v/v), 10 mM imidazole, and 0.2% DDM (m/v). The Ni-NTA column was washed with 50 mM Tris-HCl pH 8.0, 0.2% DDM (m/v) with containing 0.5 M NaCl, then 30 mM imidazole, 40 mM imidazole and the protein is eluted with 250 mM imidazole. The eluted protein was desalted using PD10 columns (PD10 -GE Healthcare Life Sciences) which were equilibrated with 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 10% glycerol (v/v), and 0.2% DDM (m/v) and diluted four times with 50 mM Tris-HCl pH 8.0, 100 mM NaCl, and 5% glycerol. The solution was mixed with a homemade preparation of B. subtilis lipids solubilized in Triton X-100 with a molar ratio of 10:1 and incubated for one hour. The quantity of lipid mix was adjusted at a lipid-to-protein ratio (LPR, in m:m) of 0.5. The DDM and Triton X-100 were removed using dialysis with Bio-beads (BioRad). The protein solution was dialyzed against 50 mM Tris-HCl pH 8.0, 100 mM NaCl, and 5% glycerol during 9 days.
For DnaB, the protein was purified by heparin-agarose affinity chromatography (5 mL HiTrap Heparin HP column from GE Healthcare Life Sciences) equilibrated with 10 mM phosphate pH 7.5, 2 mM βME.
Fractions containing the protein were pooled and loaded onto an anion exchange chromatography (5 mL HiTrap Q HP column from GE Healthcare Life Sciences). The purified protein was concentrated up to 30 mg mL −1 by centrifugation in buffer A (2.5 mM sodium phosphate, pH 7.5, 130 mM NaCl).

Preparation of the DnaB+ADP+Vi and BmrA+ADP+Vi
For the preparation of the Protein+ADP+Mg+Vi (NMR samples) or Protein+ADP+Mn+Vi (EPR samples) complexes, the protein were incubated with 1 mM Na3VO4, then 10 mM ATP (BmrA) or 10 mM ADP (DnaB) and 10 mM Mg 2+ or 1 mM Mn 2+ during 1 hour at 4°C. The preparation of vanadate solution was carefully done as described in the literature [134].

Preparation of the DnaB+ADP+AlFX and BmrA+ADP+AlFX
DnaB or BmrA was mixed with 5 mM MgCl2 and consecutively 6 mM of an NH4AlF4 solution (prepared by incubating 1 M AlCl3 solution with a 5-fold excess of 1 M NH4F solution (compared to AlCl3) for 5 min. in H2O) and 5 mM ATP (BmrA) or ADP (DnaB) and incubated for 2 hours at 4 °C.

Preparation of DnaB+nucleotide+DNA
DnaB:nucleotide complexes were prepared as described above 1 mM of (dT)20 was added to the complexes and reacted for 30 min at room temperature.

Solid-state NMR experiments
For solid-state NMR, the protein solutions were sedimented in the MAS-NMR rotor (16 h at 4 °C at 210,000 × g for DnaB, 1 h at 4 °C at 210,000 × g for BmrA) using home-build rotor filling tools [15].

EPR experiments
For EPR experiments, DnaB was concentrated to 48 mg/ml (850 μM) using a Vivaspin 500 centrifugal filter with a cut-off of 30 kDa. Then, the concentrated protein was incubated in presence of ADP 6 mM, Mn 2+ 170 μM and vanadate 7 mM 2h at 4°C. After 2h, glycerol was added to a concentration of 20 %.
For BmrA, the protein was not reconstituted in B. subtilis lipids. The protein in 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 10% glycerol (v/v), and 0.2% DDM (m/v) was concentrated to 18 mg/mL (270 μM) using ab Amicon Ultra Centrifugal filter with a cut-off of 50kDa. Then, the concentrated protein is incubated in presence of ATP 900 μM, Mn 2+ 150 μM and/or Vanadate 400 μM.
2-3 µl of protein or background solution were transferred into an 0.9 mm OD quartz capillary and flash frozen in liquid nitrogen before insertion into the spectrometer. All experiments were conducted on a Bruker Elexsys E680 X-/W-band spectrometer using a EN 680-1021H resonator. The temperature was controlled with a Helium-flow cryostat (ER 4118 CF, Oxford Instruments) and generally set to 10 K.
All measurements were conducted at W-band frequencies (~94 GHz, corresponding to ~145 MHz proton Larmor frequency). The shot repetition time was 1 ms.
Electron-electron double resonance (ELDOR)-detected NMR spectra were acquired with the echodetected hole-burning sequence tHTA -Ttp -τ -2tp -τecho, with tHTA= 50 µs, T = 10 µs, tp = 100 ns, τ = 1400 ns and an integration window of 1400 ns. The frequency of the high-turning angle (HTA) pulse was incremented in steps of 0.1 MHz over the measured range. A +/-phase cycle on the first π/2 pulse of the echo was used to eliminate unwanted coherence transfer pathways. The power of the HTA pulse, generated by the ELDOR channel of the spectrometer, was optimized such that the observed lines were as intense as possible without broadening them. The nutation frequency ν1 at the center of the resonator was about 6 MHz. The settings were held constant between protein samples and the corresponding control. Yet it is important to note that exact reproducibility of peak intensities between runs may be difficult with the resonator used because the resonator profile strongly affects line intensities in EDNMR, and hence a careful experimental setup is required.
Raw EDNMR data were background corrected with a Lorentzian line that was fitted to the central hole, and normalized to the signal intensity far off-resonance, i.e. the peak intensity corresponds to the relative hole depth. Figure S1: ATP-γ-S is hydrolysed by DnaB during MAS rotor filling. 31 P, 1 H CPMAS spectra of DnaB:ADP and DnaB:ATP-γ-S recorded directly after filling the MAS rotor over night.