Investigation of the DsbA mechanism through the synthesis and analysis of an irreversible enzyme-ligand complex.
Approaching the molecular mechanism of some enzymes is hindered by the difficulty of obtaining suitable protein-ligand complexes for structural characterization. DsbA, the major disulfide oxidase in the bacterial periplasm, is such an enzyme. Its structure has been well characterized in both its oxidized and its reduced states, but structural data about DsbA-peptide complexes are still missing. We report herein an original, straightforward, and versatile strategy for making a stable covalent complex with a cysteine-homoalanine thioether bond instead of the labile cystine disulfide bond which normally forms between the enzyme and polypeptides during the catalytic cycle of DsbA. We substituted a bromohomoalanine for the cysteine in a model 14-mer peptide derived from DsbB (PID-Br), the membrane partner of DsbA. When incubated in the presence of the enzyme, a selective nucleophilic substitution of the bromine by the thiolate of the DsbA Cys(30) occurred. The major advantage of this strategy is that it enables the direct use of the wild-type form of the enzyme, which is the most relevant to obtain unbiased information on the enzymatic mechanism. Numerous intermolecular NOEs between DsbA and PID could be observed by NMR, indicating the presence of preferential noncovalent interactions between the two partners. The thermodynamic properties of the DsbA-PID complex were measured by differential scanning calorimetry. In the complex, the values for both denaturation temperature and variation in enthalpy associated with thermal unfolding were between those of oxidized and reduced forms of DsbA. This progressive increase in stability along the DsbA catalytic pathway strongly supports the model of a thermodynamically driven mechanism.
Couprie
J
J
CEA/Saclay, Département d'Ingénierie et d'Etudes des Protéines, Gif-sur-Yvette, France.
Vinci
F
F
Dugave
C
C
Quéméneur
E
E
Moutiez
M
M
D000478:Alkylation; D001426:Bacterial Proteins / Q000737:chemistry*; D017605:Bromine Compounds / Q000737:chemistry; D002152:Calorimetry, Differential Scanning; D004926:Escherichia coli; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D010446:Peptide Fragments / Q000138:chemical synthesis; D019897:Periplasm / Q000201:enzymology; D019704:Protein Disulfide-Isomerases / Q000737:chemistry*; D013440:Sulfides / Q000737:chemistry; D013816:Thermodynamics
D001426:Bacterial Proteins; D017605:Bromine Compounds; D008024:Ligands; D010446:Peptide Fragments; D013440:Sulfides; D019704:Protein Disulfide-Isomerases