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
3DKS 2008-06-25T00:00:00+0000 1.9 DsbA substrate complex
1A23 1998-01-15T00:00:00+0000 0 SOLUTION NMR STRUCTURE OF REDUCED DSBA FROM ESCHERICHIA COLI, MINIMIZED AVERAGE STRUCTURE
1A24 1998-01-15T00:00:00+0000 0 SOLUTION NMR STRUCTURE OF REDUCED DSBA FROM ESCHERICHIA COLI, FAMILY OF 20 STRUCTURES
1A2J 1998-01-06T00:00:00+0000 2.0 OXIDIZED DSBA CRYSTAL FORM II
1A2L 1998-01-06T00:00:00+0000 2.7 REDUCED DSBA AT 2.7 ANGSTROMS RESOLUTION
1A2M 1998-01-06T00:00:00+0000 2.7 OXIDIZED DSBA AT 2.7 ANGSTROMS RESOLUTION, CRYSTAL FORM III
1AC1 1997-02-10T00:00:00+0000 2.0 DSBA MUTANT H32L
1ACV 1997-02-10T00:00:00+0000 1.9 DSBA MUTANT H32S
1BQ7 1998-08-21T00:00:00+0000 2.8 DSBA MUTANT P151A, ROLE OF THE CIS-PROLINE IN THE ACTIVE SITE OF DSBA
1DSB 1993-05-24T00:00:00+0000 2.0 CRYSTAL STRUCTURE OF THE DSBA PROTEIN REQUIRED FOR DISULPHIDE BOND FORMATION IN VIVO

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