Replacement of the active-site cysteine residues of DsbA, a protein required for disulfide bond formation in vivo.


Abstract

DsbA is a periplasmic protein of Escherichia coli that was identified genetically as being involved in the formation of disulfide bonds in secreted proteins. Its active site contains one accessible and one buried cysteine residue, separated in the primary structure by only two other residues. These cysteine residues can form a very unstable disulfide bond that is 10(3)-fold more reactive toward thiols than normal. Moreover, the mixed disulfide between the accessible cysteine residue and glutathione is 10(4)-fold more reactive than normal. Site-directed mutagenesis was carried out to replace either one or both cysteine residues by serine. Cys30 is shown to be the accessible thiol, while Cys33 is shielded from the solvent. Even though the thiol group of Cys30 is exposed and reactive, it formed a very unstable mixed disulfide with glutathione. This disulfide bond was 2.17 +/- 0.02 kcal mol-1 less stable in the native conformation than when DsbA was unfolded. If the native conformation destabilizes the mixed disulfide, the mixed disulfide must destabilize the folded conformation to the same extent. This was confirmed by demonstrating that the folded conformation of the mixed disulfide form of the mutant DsbA was 2.7 +/- 0.9 kcal mol-1 less stable than that of the reduced form; these stability effects originated almost exclusively in the folded conformation. Replacing the cysteine residues by serine destabilized the folded conformation of the reduced protein to varying extents. This suggests that the thiol groups are involved in interactions that stabilize the folded conformation, which would cause any disulfide bonds, either inter- or intramolecular, that involve these groups to be unstable. Study holds ProTherm entries: 4653, 4654, 4655, 4656 Extra Details: additive : EDTA(1 mM), periplasmic protein; cysteine; disulfide bond; thiol groups;,folded conformation

Submission Details

ID: oRKEgvxv3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:26 p.m.

Version: 1

Publication Details
Zapun A;Cooper L;Creighton TE,Biochemistry (1994) Replacement of the active-site cysteine residues of DsbA, a protein required for disulfide bond formation in vivo. PMID:8110795
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
2LEG 2011-10-26 Membrane protein complex DsbB-DsbA structure by joint calculations with solid-state NMR and X-ray experimental data
1A24 1998-09-16 SOLUTION NMR STRUCTURE OF REDUCED DSBA FROM ESCHERICHIA COLI, FAMILY OF 20 STRUCTURES
2NDO 2017-02-08 Structure of EcDsbA-sulfonamide1 complex
1A23 1998-09-16 SOLUTION NMR STRUCTURE OF REDUCED DSBA FROM ESCHERICHIA COLI, MINIMIZED AVERAGE STRUCTURE
1FVK 1997-08-20 1.7 THE 1.7 ANGSTROM STRUCTURE OF WILD TYPE DISULFIDE BOND FORMATION PROTEIN (DSBA)
4ZIJ 2016-05-11 1.78 Crystal structure of E.Coli DsbA in complex with 2-(4-iodophenylsulfonamido) benzoic acid
1ACV 1997-10-15 1.9 DSBA MUTANT H32S
3DKS 2009-05-12 1.9 DsbA substrate complex
2B3S 2006-09-05 1.96 structure of the DSBA mutant (P31G-C33A)
6BR4 2017-12-27 1.99 Crystal structure of Escherichia coli DsbA in complex with {N}-methyl-1-(3-thiophen-2-ylphenyl)methanamine
6BQX 2017-12-27 1.99 Crystal structure of Escherichia coli DsbA in complex with N-methyl-1-(4-phenoxyphenyl)methanamine
1DSB 1994-01-31 2.0 CRYSTAL STRUCTURE OF THE DSBA PROTEIN REQUIRED FOR DISULPHIDE BOND FORMATION IN VIVO
1AC1 1997-10-15 2.0 DSBA MUTANT H32L
1U3A 2005-05-03 2.0 mutant DsbA
1A2J 1998-09-16 2.0 OXIDIZED DSBA CRYSTAL FORM II
1FVJ 1997-05-15 2.06 THE 2.06 ANGSTROM STRUCTURE OF THE H32Y MUTANT OF THE DISULFIDE BOND FORMATION PROTEIN (DSBA)
1UN2 2003-09-26 2.4 Crystal structure of circularly permuted CPDSBA_Q100T99: Preserved Global Fold and Local Structural Adjustments
4TKY 2015-01-14 2.5 The complex structure of E. coli DsbA bound to a peptide at the DsbA/DsbB interface
1TI1 2005-05-03 2.6 crystal structure of a mutant DsbA
2B6M 2006-10-17 2.65 Structure of the DsbA mutant (P31A-C33A)
1A2L 1998-07-08 2.7 REDUCED DSBA AT 2.7 ANGSTROMS RESOLUTION
1A2M 1998-07-08 2.7 OXIDIZED DSBA AT 2.7 ANGSTROMS RESOLUTION, CRYSTAL FORM III
1BQ7 1999-08-20 2.8 DSBA MUTANT P151A, ROLE OF THE CIS-PROLINE IN THE ACTIVE SITE OF DSBA
2ZUP 2009-04-14 3.7 Updated crystal structure of DsbB-DsbA complex from E. coli
2HI7 2006-12-05 3.7 Crystal structure of DsbA-DsbB-ubiquinone complex
3E9J 2008-11-25 3.7 Structure of the charge-transfer intermediate of the transmembrane redox catalyst DsbB

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
99.5 Thiol:disulfide interchange protein DsbA P52235 DSBA_SHIFL
99.5 Thiol:disulfide interchange protein DsbA P0A4L5 DSBA_ECOL6
99.5 Thiol:disulfide interchange protein DsbA P0A4L6 DSBA_ECO27
100.0 Thiol:disulfide interchange protein DsbA P0AEG4 DSBA_ECOLI
100.0 Thiol:disulfide interchange protein DsbA P0AEG5 DSBA_ECO57