Competition between DsbA-mediated oxidation and conformational folding of RTEM1 beta-lactamase.


Abstract

Similar to other proteins of the periplasm of Escherichia coli, TEM 1 beta-lactamase contains only a single disulfide bond. It can fold to its native conformation in both the presence and the absence of this disulfide bond. The GdmC1-dependent equilibrium unfolding of beta-lactamase in vitro is well described by a N reversible I reversible U three-state model in which the native protein (N) first reacts to an intermediate of the molten globule type (I) and then to the unfolded state (U). We find that the disulfide bond of beta-lactamase stabilizes I relative to U, but does not change the stability of N relative to I. The I reversible U transition is an extremely rapid reaction for both reduced and oxidized beta-lactamase, but the N reversible I folding kinetics are slow and identical in the presence and the absence of the disulfide bond. This insensitivity of the N reversible I equilibrium and kinetics suggests that the region around the disulfide bond is already native-like folded and is presumably buried in the intermediate I, prior to the slow and rate-limiting events of folding. This was confirmed by measuring the stability of the disulfide bond, which, to a first approximation, is identical in N and I. In native, reduced beta-lactamase, the thiol groups are inaccessible for oxidation by DsbA, but at the stage of the molten globule intermediate I oxidation is still possible, because I is in fast exchange with the unfolded protein U. The introduction of the disulfide bond into beta-lactamase by DsbA competes with conformational folding at the stage of the final slow steps in the folding of the reduced protein. The major problem in the oxidation of proteins with one or two disulfide bonds (such as beta-lactamase) is not the formation of incorrect disulfide bonds, but the premature burial of the thiol groups by the rapid conformational folding of the reduced protein. DsbA, the major thiol/ disulfide isomerase of the bacterial periplasm, meets this problem. It is a very strong oxidant, and its reaction with cysteine residues in unfolded proteins is extremely fast. Study holds ProTherm entries: 4842, 4843, 4844, 4845 Extra Details: additive : EDTA(2 mM),N -> I disulfide bond; native conformation; molten globule;,thiol groups

Submission Details

ID: SHqF9tzQ3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:27 p.m.

Version: 1

Publication Details
Frech C;Wunderlich M;Glockshuber R;Schmid FX,Biochemistry (1996) Competition between DsbA-mediated oxidation and conformational folding of RTEM1 beta-lactamase. PMID:8784194
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
2FFY 2006-03-28 1.07 AmpC beta-lactamase N289A mutant in complex with a boronic acid deacylation transition state analog compound SM3
2HDS 2006-11-07 1.16 AmpC beta-lactamase in complex with 4-Methanesulfonylamino benzoic acid
4KZ5 2014-05-21 1.35 Crystal structure of AmpC beta-lactamase in complex with fragment 5 (N-{[3-(2-chlorophenyl)-5-methyl-1,2-oxazol-4-yl]carbonyl}glycine)
4KZB 2014-05-21 1.37 Crystal structure of AmpC beta-lactamase in complex with fragment 50 (N-(methylsulfonyl)-N-phenyl-alanine)
4OKP 2014-05-28 1.37 Crystal structure of AmpC beta-lactamase in complex with the product form of 7-amino-desacetoxycephalosporanic acid
1PI4 2004-02-24 1.39 Structure of N289A mutant of AmpC in complex with SM3, a phenylglyclboronic acid bearing the cephalothin R1 side chain
4LV3 2014-07-30 1.42 AmpC beta-lactamase in complex with (3,5-di-tert-butylphenyl) boronic acid
4KZ4 2014-05-21 1.42 Crystal structure of AmpC beta-lactamase in complex with fragment 60 (2-[(propylsulfonyl)amino]benzoic acid)
4KZ7 2014-05-21 1.43 Crystal structure of AmpC beta-lactamase in complex with fragment 16 ((1R,4S)-4,7,7-trimethyl-3-oxo-2-oxabicyclo[2.2.1]heptane-1-carboxylic acid)
4E3L 2012-09-26 1.43 Crystal structure of AmpC beta-lactamase in complex with a 3-chloro-4-tetrazolyl benzene sulfonamide boronic acid inhibitor
4E3K 2012-09-26 1.43 Crystal structure of AmpC beta-lactamase in complex with a designed 4-tetrazolyl pyridine sulfonamide boronic acid inhibitor
4E3M 2012-09-26 1.44 Crystal structure of AmpC beta-lactamase in complex with a 2-chloro-4-tetrazolyl benzene sulfonamide boronic acid inhibitor
4OLD 2014-05-28 1.48 Crystal structure of AmpC beta-lactamase in complex with the product form of (6R,7R)-7-amino-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid
2HDU 2006-11-07 1.49 AmpC beta-lactamase in complex with 2-acetamidothiophene-3-carboxylic acid
1PI5 2004-02-24 1.49 Structure of N289A mutant of AmpC in complex with SM2, carboxyphenylglycylboronic acid bearing the cephalothin R1 side chain
4E3N 2012-09-26 1.49 Crystal structure of AmpC beta-lactamase in complex with a 2-trifluoromethyl-4-tetrazolyl benzene sulfonamide boronic acid inhibitor
1L0G 2002-08-14 1.5 X-ray Crystal Structure of AmpC S64G Mutant beta-Lactamase
3FKW 2009-03-24 1.5 AmpC K67R mutant apo structure
6DPZ 2018-07-04 1.5 X-ray crystal structure of AmpC beta-lactamase with inhibitor
1KVL 2002-03-13 1.53 X-ray Crystal Structure of AmpC S64G Mutant beta-Lactamase in Complex with Substrate and Product Forms of Cephalothin
1L0D 2002-08-14 1.53 X-ray Crystal Structure of AmpC S64D Mutant beta-Lactamase
4E3O 2012-09-26 1.6 Crystal structure of AmpC beta-lactamase in complex with a small chloromethyl sulfonamide boronic acid inhibitor
4E3I 2012-09-26 1.6 Crystal structure of AmpC beta-lactamase in complex with a designed 3-carboxyl benzyl sulfonamide boronic acid inhibitor
3O86 2010-11-03 1.6 Crystal structure of AmpC beta-lactamase in complex with a sulfonamide boronic acid inhibitor
4KZA 2014-05-21 1.6 Crystal structure of AmpC beta-lactamase in complex with fragment 48 (3-(cyclopropylsulfamoyl)thiophene-2-carboxylic acid)
3IXB 2010-02-16 1.63 X-ray crystal structure of the extended-spectrum AmpC E219K mutant beta-lactamase complexed with benzo(b)thiophene-2-boronic acid (BZB) at 1.63 Angstrom resolution
3IWI 2010-02-16 1.64 X-ray crystal structure of the extended-spectrum AmpC omega loop insertion (H210AAA) mutant beta-lactamase at 1.64 Angstrom resolution
3O88 2010-11-03 1.64 Crystal structure of AmpC beta-lactamase in complex with a sulfonamide boronic acid inhibitor
4LV0 2014-07-30 1.65 AmpC beta-lactamase in complex with m-aminophenyl boronic acid
4LV2 2014-07-30 1.65 AmpC beta-lactamase in complex with [1-(6-chloropyrimidin-4-yl)-1H-pyrazol-4-yl] boronic acid
4JXG 2014-10-29 1.65 Crystal Structure of AmpC beta-lactamase from E. coli in Complex with Oxacillin
1L0F 2002-08-14 1.66 X-ray Crystal Structure of AmpC N152H Mutant beta-Lactamase
4KZ3 2014-05-21 1.67 Crystal structure of AmpC beta-lactamase in complex with fragment 44 (5-chloro-3-sulfamoylthiophene-2-carboxylic acid)
4KZ6 2014-05-21 1.68 Crystal structure of AmpC beta-lactamase in complex with fragment 13 ((2R,6R)-6-methyl-1-(3-sulfanylpropanoyl)piperidine-2-carboxylic acid)
4OLG 2014-05-28 1.71 Crystal structure of AmpC beta-lactamase in complex with covalently bound N-formyl 7-aminocephalosporanic acid
1O07 2003-08-26 1.71 Crystal Structure of the complex between Q120L/Y150E mutant of AmpC and a beta-lactam inhibitor (MXG)
1KE4 2002-07-17 1.72 X-ray crystal structure of AmpC beta-lactamase from E. coli
1MY8 2003-03-04 1.72 AmpC beta-lactamase in complex with an M.carboxyphenylglycylboronic acid bearing the cephalothin R1 side chain
4KZ9 2014-05-21 1.72 Crystal structure of AmpC beta-lactamase in complex with fragment 41 ((4R,4aS,8aS)-4-phenyldecahydroquinolin-4-ol)
1LLB 2002-10-02 1.72 Crystal Structure Of AmpC beta-Lactamase From E. Coli In Complex With ATMO-penicillin
4LV1 2014-07-30 1.74 AmpC beta-lactamase in complex with [1-(3-chlorophenyl)-1H-pyrazol-4-yl] boronic acid
4KG6 2014-10-29 1.75 Crystal Structure of AmpC beta-lactamase N152G Mutant from E. coli
5JOC 2017-05-03 1.75 Crystal structure of the S61A mutant of AmpC BER
1FSY 2001-03-14 1.75 AMPC BETA-LACTAMASE FROM E. COLI COMPLEXED WITH INHIBITOR CLOXACILLINBORONIC ACID
4JXV 2014-04-30 1.76 X-ray crystal structure of AmpC beta-lactamase from E. coli in complex with a non-covalent inhibitor 3-{[2-(4-CARBOXYPHENYL)ETHYL]SULFAMOYL}THIOPHENE-2-CARBOXYLIC ACID (compound 5)
3O87 2010-11-03 1.78 Crystal structure of AmpC beta-lactamase in complex with a sulfonamide boronic acid inhibitor
6DPT 2018-07-04 1.79 X-ray crystal structure of AmpC beta-lactamase with nanomolar inhibitor
2P9V 2007-08-14 1.8 Structure of AmpC beta-lactamase with cross-linked active site after exposure to small molecule inhibitor
2R9W 2008-04-15 1.8 AmpC beta-lactamase with bound Phthalamide inhibitor
3GR2 2009-04-14 1.8 AmpC beta-lactamase in complex with Fragment-based Inhibitor
3GQZ 2009-04-14 1.8 AmpC beta-lactamase in complex with Fragment-based Inhibitor
3GV9 2009-04-14 1.8 AmpC beta-lactamase in complex with Fragment-based Inhibitor
3GVB 2009-04-14 1.8 AmpC beta-lactamase in complex with Fragment-based Inhibitor
1LL5 2002-11-27 1.8 X-ray crystal structure of AmpC WT beta-lactamase in complex with covalently bound imipenem
4E3J 2012-09-26 1.8 Crystal structure of AmpC beta-lactamase in complex with a designed 4-tetrazolyl benzene sulfonamide boronic acid inhibitor
1MXO 2003-03-04 1.83 AmpC beta-lactamase in complex with an m.carboxyphenylglycylboronic acid bearing the cephalothin R1 side chain
1I5Q 2001-06-20 1.83 CRYSTAL STRUCTURE OF THE E. COLI AMPC BETA-LACTAMASE MUTANT N152A COVALENTLY ACYLATED WITH THE INHIBITORY BETA-LACTAM, MOXALACTAM
3IWQ 2010-02-16 1.84 X-ray crystal structure of the extended-spectrum AmpC E219K mutant beta-lactamase at 1.84 Angstrom resolution
3FKV 2009-03-24 1.85 AmpC K67R mutant complexed with benzo(b)thiophene-2-boronic acid (bzb)
2PU2 2008-04-15 1.86 AmpC beta-lactamase with bound Phthalamide inhibitor
1LL9 2002-10-02 1.87 Crystal Structure Of AmpC beta-Lactamase From E. Coli In Complex With Amoxicillin
4KG2 2014-10-29 1.89 Crystal Structure of AmpC beta-lactamase from E. coli in Complex with Cefotaxime
4KEN 2014-10-29 1.89 Crystal Structure of AmpC beta-lactamase N152G Mutant in Complex with Cefoxitin
1L0E 2002-08-14 1.9 X-ray Crystal Structure of AmpC K67Q Mutant beta-Lactamase
3GTC 2009-04-14 1.9 AmpC beta-lactamase in complex with Fragment-based Inhibitor
4JXS 2014-05-07 1.9 X-ray crystal structure of AmpC beta-lactamase from E. coli in complex with a non-covalent inhibitor 3-[(4-CARBOXYBENZYL)SULFAMOYL]THIOPHENE-2-CARBOXYLIC ACID (compound 4)
6DPX 2018-07-04 1.9 X-ray crystal structure of AmpC beta-lactamase with inhibitor
2R9X 2008-04-15 1.9 AmpC beta-lactamase with bound Phthalamide inhibitor
1FSW 2001-03-14 1.9 AMPC BETA-LACTAMASE FROM E. COLI COMPLEXED WITH INHIBITOR CEPHALOTHINBORONIC ACID
3IWO 2010-02-16 1.9 X-ray crystal structure of the extended-spectrum AmpC Y221G mutant beta-lactamase at 1.90 Angstrom resolution
6DPY 2018-07-04 1.91 X-ray crystal structure of AmpC beta-lactamase with inhibitor
1L2S 2002-07-17 1.94 X-ray crystal structure of AmpC beta-lactamase from E. coli in complex with a DOCK-predicted non-covalent inhibitor
1XGI 2005-05-03 1.96 AmpC beta-lactamase in complex with 3-(3-nitro-phenylsulfamoyl)-thiophene-2-carboxylic acid
1XGJ 2005-05-03 1.97 AmpC beta-lactamase in complex with 3-(4-carboxy-2-hydroxy-phenylsulfamoyl)-thiophene-2-carboxylic acid
2PU4 2008-04-15 2.0 AmpC beta-lacamase with bound covalent oxadiazole inhibitor
2BLS 1998-08-12 2.0 AMPC BETA-LACTAMASE FROM ESCHERICHIA COLI
2RCX 2007-11-27 2.0 AmpC Beta-lactamase in complex with (1R)-1-(2-Thiophen-2-yl-acetylamino)-1-(3-(2-carboxyvinyl)-phenyl) methylboronic acid
1IEL 2001-08-15 2.0 Crystal Structure of AmpC beta-lactamase from E. coli in Complex with Ceftazidime
1KVM 2002-03-13 2.06 X-ray Crystal Structure of AmpC WT beta-Lactamase in Complex with Covalently Bound Cephalothin
1GA9 2001-07-25 2.1 CRYSTAL STRUCTURE OF AMPC BETA-LACTAMASE FROM E. COLI COMPLEXED WITH NON-BETA-LACTAMASE INHIBITOR (2, 3-(4-BENZENESULFONYL-THIOPHENE-2-SULFONYLAMINO)-PHENYLBORONIC ACID)
3BM6 2009-02-17 2.1 AmpC beta-lactamase in complex with a p.carboxyphenylboronic acid
2HDQ 2006-11-07 2.1 AmpC beta-lactamase in complex with 2-carboxythiophene
3GSG 2009-04-14 2.1 AmpC beta-lactamase in complex with Fragment-based Inhibitor
4KG5 2014-10-29 2.11 Crystal Structure of AmpC beta-lactamase N152G Mutant in Complex with Cefotaxime
3IXG 2010-02-16 2.14 X-ray crystal structure of the extended-spectrum AmpC T70I mutant beta-lactamase with and without benzo(b)thiophene-2-boronic acid bound at 2.14 Angstrom resolution
1KE3 2002-07-17 2.15 X-ray crystal structure of AmpC beta-lactamase from E. coli in complex with the inhibitor 4,4'-biphenyldiboronic acid
1KDS 2002-07-17 2.15 X-ray crystal structure of AmpC beta-lactamase from E. coli in complex with the inhibitor 3-nitrophenylboronic acid
2HDR 2006-11-07 2.2 AmpC beta-lactamase in complex with 4-Amino-3-hydroxybenzoic acid
1FCO 2000-12-04 2.2 CRYSTAL STRUCTURE OF THE E. COLI AMPC BETA-LACTAMASE COVALENTLY ACYLATED WITH THE INHIBITORY BETA-LACTAM, MOXALACTAM
2I72 2007-09-11 2.2 AmpC beta-lactamase in complex with 5-diformylaminomethyl-benzo[b]thiophen-2-boronic acid
1C3B 1999-11-24 2.25 AMPC BETA-LACTAMASE FROM E. COLI COMPLEXED WITH INHIBITOR, BENZO(B)THIOPHENE-2-BORONIC ACID (BZB)
4KZ8 2014-05-21 2.28 Crystal structure of AmpC beta-lactamase in complex with fragment 20 (1,3-diethyl-2-thioxodihydropyrimidine-4,6(1H,5H)-dione)
1KDW 2002-07-17 2.28 X-ray crystal structure of AmpC beta-lactamase from E. coli in complex with the inhibitor 4-carboxyphenylboronic acid
1KE0 2002-07-17 2.3 X-ray crystal structure of AmpC beta-lactamase from E. coli in complex with the inhibitor 4-(carboxyvin-2-yl)phenylboronic acid
1IEM 2001-08-15 2.3 Crystal Structure of AmpC beta-lactamase from E. coli in Complex with a Boronic Acid Inhibitor (1, CefB4)
4JXW 2014-05-07 2.3 X-ray crystal structure of AmpC beta-lactamase from E. coli in complex with a non-covalent inhibitor 3-{[3-(4-CARBOXYPHENYL)PROPYL]SULFAMOYL}THIOPHENE-2-CARBOXYLIC ACID (compound 6)
3IXH 2010-02-16 2.3 X-ray crystal structure of the extended-spectrum AmpC Y221G mutant beta-lactamase in complex with cefotaxime at 2.3 Angstrom resolution
3BLS 1998-08-12 2.3 AMPC BETA-LACTAMASE FROM ESCHERICHIA COLI
1FCN 2000-12-04 2.35 Crystal Structure of the E. Coli AMPC Beta-Lactamase Mutant Q120L/Y150E Covalently Acylated with the Substrate Beta-Lactam LORACARBEF
1FCM 2000-12-04 2.46 CRYSTAL STRUCTURE OF THE E.COLI AMPC BETA-LACTAMASE MUTANT Q120L/Y150E COVALENTLY ACYLATED WITH THE INHIBITORY BETA-LACTAM, CLOXACILLIN
3GRJ 2009-04-14 2.49 AmpC beta-lactamase in complex with Fragment-based Inhibitor
3IXD 2010-02-16 2.64 X-ray crystal structure of the extended-spectrum AmpC V298E mutant beta-lactamase at 2.64 Angstrom resolution

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Beta-lactamase P00811 AMPC_ECOLI