Structural analysis of three His32 mutants of DsbA: support for an electrostatic role of His32 in DsbA stability.


Abstract

DsbA, a 21-kDa protein from Escherichia coli, is a potent oxidizing disulfide catalyst required for disulfide bond formation in secreted proteins. The active site of DsbA is similar to that of mammalian protein disulfide isomerases, and includes a reversible disulfide bond formed from cysteines separated by two residues (Cys30-Pro31-His32-Cys33). Unlike most protein disulfides, the active-site disulfide of DsbA is highly reactive and the oxidized form of DsbA is much less stable than the reduced form at physiological pH. His32, one of the two residues between the active-site cysteines, is critical to the oxidizing power of DsbA and to the relative instability of the protein in the oxidized form. Mutation of this single residue to tyrosine, serine, or leucine results in a significant increase in stability (of approximately 5-7 kcal/mol) of the oxidized His32 variants relative to the oxidized wild-type protein. Despite the dramatic changes in stability, the structures of all three oxidized DsbA His32 variants are very similar to the wild-type oxidized structure, including conservation of solvent atoms near the active-site residue, Cys30. These results show that the His32 residue does not exert a conformational effect on the structure of DsbA. The destabilizing effect of His32 on oxidized DsbA is therefore most likely electrostatic in nature. Study holds ProTherm entries: 8910, 8911, 8912, 8913, 8914, 8915, 8916, 8917, 14482, 14483, 14484, 14485, 14486, 14487, 14488 Extra Details: additive : EDTA(1 mM), DsbA; mutagenesis; oxidoreductase; protein crystallography;,protein stability; thioredoxin fold

Submission Details

ID: LbyEAxQd

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:37 p.m.

Version: 1

Publication Details
Guddat LW;Bardwell JC;Glockshuber R;Huber-Wunderlich M;Zander T;Martin JL,Protein Sci. (1997) Structural analysis of three His32 mutants of DsbA: support for an electrostatic role of His32 in DsbA stability. PMID:9300489
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
1A24 1998-09-16 SOLUTION NMR STRUCTURE OF REDUCED DSBA FROM ESCHERICHIA COLI, FAMILY OF 20 STRUCTURES
2LEG 2011-10-26 Membrane protein complex DsbB-DsbA structure by joint calculations with solid-state NMR and X-ray experimental data
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
6POH 2019-11-06 1.67 Crystal Structure of EcDsbA in complex alkyl ether 21
1FVK 1997-08-20 1.7 THE 1.7 ANGSTROM STRUCTURE OF WILD TYPE DISULFIDE BOND FORMATION PROTEIN (DSBA)
6PVY 2019-12-25 1.74 E.coli DsbA in complex with benzofuran compound 26 ([6-(3-methoxyphenoxy)-1-benzofuran-3-yl]acetic acid)
6POI 2019-11-06 1.77 Crystal Structure of EcDsbA in complex phenyl ether 25
4ZIJ 2016-05-11 1.78 Crystal structure of E.Coli DsbA in complex with 2-(4-iodophenylsulfonamido) benzoic acid
6POQ 2019-11-06 1.8 Crystal Structure of EcDsbA in complex with anisidine 16
1ACV 1997-10-15 1.9 DSBA MUTANT H32S
3DKS 2009-05-12 1.9 DsbA substrate complex
6PMF 2019-11-06 1.95 Crystal Structure of EcDsbA in complex with aniline 15
2B3S 2006-09-05 1.96 structure of the DSBA mutant (P31G-C33A)
6BQX 2017-12-27 1.99 Crystal structure of Escherichia coli DsbA in complex with N-methyl-1-(4-phenoxyphenyl)methanamine
6PVZ 2019-12-25 1.99 E.coli DsbA in complex with benzofuran compound 28 ((6-benzyl-1-benzofuran-3-yl)acetic acid)
6BR4 2017-12-27 1.99 Crystal structure of Escherichia coli DsbA in complex with {N}-methyl-1-(3-thiophen-2-ylphenyl)methanamine
6PML 2019-11-06 2.0 Crystal Structure of EcDsbA in complex benzyl ether 23
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
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
2ZUP 2009-04-14 3.7 Updated crystal structure of DsbB-DsbA complex from E. coli

Relevant UniProtKB Entries

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