An active site–tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase

Citrate synthase from the thermophilic euryarchaeon T. acidophilum fused to a hexahistidine tag was purified and biochemically characterized. The structure of the unliganded enzyme at 2.2 Å resolution contains tail–active site contacts in half of the active sites.

DNA-modifying enzymes were from New England Biolabs (Beverly, MA). A Vent DNA polymerase PCR product, obtained with ODN primers 1306 and 1307 (Table 1)  and ODN 2067 (5 -ACCGCCtTGCAGCGGACCCTTG) were used to construct the TpCSH6-H222Q expression plasmid pJK511. Bidirectional Sanger sequencing of the coding region was used to confirm that both plasmids contained the expected DNA sequences.
Enzyme kinetics and inhibition analysis Enzyme activities at 298 K were recorded on a Cary 100 spectrophotometer (Varian) equipped with a Peltier temperature controller.
Nonlinear least-squares fitting was performed using using gnuplot (version 4.4).
AcMX, synthesized by Hong Jiang as described (Francois et al., 2006), was used to prepare the TpCS · CitMX complex (PDB entry 2r9e) and assessed as a TpCSH6 inhibitor.
Initial velocities were measured in a final volume of 0.7 mL that contained 50 mM potassium phosphate, pH 8.0, 100 mM KCl, 0.4 mM OAA, 0.3 mM DTNB, varied AcCoA, and several fixed concentrations of AcMX. Reactions were initiated by the addition of TpCSH6 (0.6 mg).
Lineweaver-Burk plots showed a competitive inhibition pattern. All initial velocity data were globally fit to a Michaelis-Menten equation in which K M,app = K M (1 + [AcMX]/K i ).
Ligand affinity measurements Fluorescence emission spectra (excitation 295 nm) were recorded using a Fluoromax-3 (Horiba) spectrofluorimeter at 298 K. Emission spectra were corrected for dilution and, using a control titration of N -acetyl-l-tryptophanamide, the inner filter effect due to OAA. Titration data monitoring changes in the fluorescence emission intensity at 315 nm (F ) were plotted by normalizing F by the emission intensity measured in the absence of titrant (F o ).
Protein-ligand affinities were determined by fits to Eqn. S1 (for is the subunit concentration of the enzyme) or Eqn. S2, which accounts for changes in [L] (either [OAA] or [AcMX]) due to ligand depletion.   Figure S5. AcMX affinity for TpCSH6·OAA determined by fluorescence titration. Experimental conditions were the same as described in Fig. S4, except that 0.2 mM OAA was also present. The AcMX-dependent increase in F is due to conversion of the fluorescence quencher OAA to CitMX. The solid line depicts a fit of the titration data to Eqn. S2, divided by F o , with K d = 1.5 ± 0.1 µM and maximal ∆F/F o = 1.24 ± 0.01.

Trp348
Thr346 Figure S6. Representative map-model agreement. Electron density maps near Trp348 (subunit C) are shown: σA-weighted 2mFo-DFc (grey mesh, 1.5σ) and mFo-DFc (green and red mesh, ±3σ). The Trp348 rotamer, which is essentially the same as in liganded TpCS structures, is flipped (-171 • rotation) relative to the rotamer observed in the structure of TpCS (Russell et al., 1994).  Figure S7. Primary sequence, secondary structure, and domain motions in TpCS(H6). The C-terminal appendage of TpCSH6 is indicated with a grey background. In keeping with prior practice, the initiator codon is assigned the number zero. The elements of secondary structure are given the same designations as in TpCS, apart from a short helix (α16) and Cterminal appendage helix (αU) that were not present in the apo TpCS structure. The colored symbols correspond to mobile domains (underlined) and hinge residues (carets) identified by DynDom comparisons of TpCSH6 with one ligand-bound TpCS subunit.   Figure S8. Active site of TpCS · CitMX complex (PDB entry 2r9e) showing selected polar protein-ligand interactions. CitMX differs from CitCoA by an atomic substitution (S −→ CH 2 ) indicated with an arrow. TpCS forms CitMX by condensing AcMX and OAA, forming the bond denoted in magenta. Residues with names shown in red are also involved in binding the C-terminus of TpCSH6 subunit A or D (Fig. 2). The prime denotes a residue from the partner subunit in the TpCS dimer; note that the C-terminus only engages residues from one subunit. Polar contacts with the CoA region by Leu257, Gly259, Phe260, Asn315, and Arg 361 are omitted.