Glutaredoxin 3 from Escherichia coli : NMR structure analysis and structural aspects of the enzymatic mechanism

The structure of Escherichia coli glutaredoxin 3 (Grx3) was investigated using NMR spectroscopy in combination with isotope labeling. Nearly complete sequence specific assignment were obtained for the reduced Grx3 using a 15N enriched protein sample. The secondary structure and topology of reduced Grx3 was shown to be similar to another glutaredoxin in E. coli, Grx1, based on the analysis of chemical shifts, coupling constants, amide exchange rates and short and medium range NOEs. In the NMR spectra of reduced Grx3, the thiol proton of the more C-terminal active site cysteine, Cys14, is present despite presaturation of the solvent resonance, with a chemical shift of 7.6 ppm. The large downfield shift and the fact that the thiol proton is observed in the NMR spectra suggests that it is hydrogen bonded and protected from exchange with solvent. Additionally, the sequence specific assignments for reduced, unfolded Grx3 at pH 3.5 were obtained using a combination of a 3D 15N-HSQC-(TOCSY-NOESY)-15N-HSQC and a 3D-ROESY-HSQC experiment. The former makes use of the relatively large chemical shift dispersion of the 15N chemical shift in both indirect dimensions to overcome chemical shift degeneracies present in the unfolded protein. Analysis of coupling constants and NOEs, combined with a comparison between the observed chemical shifts and published values for random coil chemical shifts, indicate that Grx3 is practically completely unfolded at this pH. A technique for producing fractionally [13C]-enriched protein was developed, using an algal hydrolysate as the sole carbon source. The fractional enrichment was utilized in an HSQC experiment for the determination of one-bond coupling constants, 1JHC. Accurate 1JHC couplings of C[alpha]H groups contain information on the dihedral angle [psi], and are thus potentially useful constraints for NMR structure determinations. The NMR structure of the Grx3-glutathione mixed disulfide (Grx3-SG) was determined using a Grx3 C14S-C65Y mutant. The Grx3-SG complex is an intermediate in the enzymatic reaction and the C14S mutation was needed to trap the intermediate. The structure was calculated with the program DYANA and energy minimized in explicit water using the program OPAL. The RMSD of protein backbone atoms (N, C[alpha], C') in a final set of 20 conformers was 0.57 Å relative to the mean structure. The interactions between Grx3 and glutathione were defined by 21 intermolecular distance constraints, and the glutathione was found to bind in an anti-parallel ß bridge to Va152 of Grx3. The structure also suggests the presence of a second glutathione binding site which could explain the observed specificity for reduction of glutaredoxin-glutathione mixed disulfides by glutathione. Except for the active site, the NMR spectra of reduced Grx3 and Grx3-SG are very similar. A comparison of chemical shifts and coupling constants in the different spectra led to a proposal of the interactions stabilizing the Cys11 thiolate in reduced Grx3, which were further supported by activity measurements for a number of Grx3 mutants

Direct NMR observation of the Cys-14 thiol proton of reduced Escherichia coli glutaredoxin-3 supports the presence of an active site thiol-thiolate hydrogen bond

AbstractThe active site of Escherichia coli glutaredoxin-3 (Grx3) consists of two redox active cysteine residues in the sequence -C11-P-Y-C14-H-. The 1H NMR resonance of the cysteine thiol proton of Cys-14 in reduced Grx3 is observed at 7.6 ppm. The large downfield shift and NOEs observed with this thiol proton resonance suggest the presence of a hydrogen bond with the Cys-11 thiolate, which is shown to have an abnormally low pKa value. A hydrogen bond would also agree with activity data of Grx3 active site mutants. Furthermore, the activity is reduced in a Grx3 H15V mutant, indicating electrostatic contributions to the stabilization of the Cys-11 thiolate