The coordination of the isomerization of a conserved non-prolyl cis peptide bond with the rate-limiting steps in the folding of dihydrofolate reductase.


Abstract

The propensity for peptide bonds to adopt the trans configuration in native and unfolded proteins, and the relatively slow rates of cis-trans isomerization reactions, imply that the formation of cis peptide bonds in native conformations are likely to limit folding reactions. The role of the conserved cis Gly95-Gly96 peptide bond in dihydrofolate reductase (DHFR) from Escherichia coli was examined by replacing Gly95 with alanine. The introduction of a beta carbon at position 95 is expected to increase the propensity for the trans isomer and perturb the isomerization reaction required to reach the native conformation. Although G95A DHFR is 1.30 kcal mol(-1) less stable than the wild-type protein, it adopts a well-folded structure that can be chemically denatured in a cooperative fashion. The mutant protein also retains the complex refolding kinetic pattern attributed to a parallel-channel mechanism in wild-type DHFR. The spectroscopic response upon refolding monitored by Trp fluorescence and the absence of a Trp/Trp exciton coupling apparent in the far-UV CD spectrum of the wild-type protein, however, indicated that the tertiary structure of the folded state for G95A DHFR is altered. The addition of methotrexate (MTX), a tight-binding inhibitor, to folded G95A DHFR restored the exciton coupling and the fluorescence properties through five slow kinetic events whose relaxation times are independent of the ligand and the denaturant concentrations. The results were interpreted to mean that MTX-binding drives the formation of the cis isomer of the peptide bond between Ala95 and Gly96 in five compact and stable but not wild-type-like conformations that contain the trans isomer. Folding studies in the presence of MTX for both wild-type and G95A DHFR support the notion that the cis peptide bond between Gly95 and Gly96 in the wild-type protein forms during four parallel rate-limiting steps, which are primarily controlled by folding reactions, and lead directly to a set of native, or native-like, conformers. The isomerization of the cis peptide bond is not a source of the parallel channels that characterize the complex folding mechanism for DHFR. Study holds ProTherm entries: 16141, 16142 Extra Details: 0.2 mM K2EDTA and 1 mM beta-mercaptoethanol were added in the experiment. non-prolyl; peptide bond; isomerization; folding; DHFR

Submission Details

ID: rSU4URTD3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:47 p.m.

Version: 1

Publication Details
Svensson AK;O'Neill JC;Matthews CR,J. Mol. Biol. (2003) The coordination of the isomerization of a conserved non-prolyl cis peptide bond with the rate-limiting steps in the folding of dihydrofolate reductase. PMID:12559923
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
4PDJ 2015-04-15 Neutron crystal Structure of E.coli Dihydrofolate Reductase complexed with folate and NADP+
6CW7 2019-01-09 1.03 E. coli DHFR product complex with (6S)-5,6,7,8-TETRAHYDROFOLATE
6CXK 2019-01-09 1.11 E. coli DHFR substrate complex with Dihydrofolate
4NX7 2014-01-15 1.15 single cryogenic temperature model of DHFR
4KJJ 2013-08-21 1.15 Cryogenic WT DHFR
5CC9 2015-08-05 1.2 L28F E.coli dihydrofolate reductase complexed with 5,10-dideazatetrahydrofolate and oxidized nicotinamide adenine dinucleotide phosphate
6CYV 2019-01-09 1.3 E. coli DHFR ternary complex with NADP and dihydrofolate
4NX6 2014-01-15 1.35 single room temperature model of DHFR
4KJK 2013-08-21 1.35 Room Temperature WT DHFR
6MR9 2019-05-15 1.35 E. coli DHFR complex with a reaction intermediate
6MT8 2019-05-15 1.35 E. coli DHFR complex modeled with two ligand states
5UII 2018-01-31 1.35 structure of DHFR with bound buformin and NADP
6MTH 2019-05-15 1.35 E. coli DHFR complex modeled with three ligand states
4KJL 2013-08-21 1.38 Room Temperature N23PPS148A DHFR
5W3Q 2017-08-09 1.4 L28F E.coli DHFR in complex with NADPH
5CCC 2015-08-05 1.5 wild-type E.coli dihydrofolate reductase complexed with 5,10-dideazatetrahydrofolate and oxidized nicotinamide adenine dinucleotide phosphate
3DAU 2009-04-14 1.5 Crystal structure of the ternary MTX NADPH complex of Escherichia coli dihydrofolate reductase
1RA9 1996-12-23 1.55 DIHYDROFOLATE REDUCTASE COMPLEXED WITH NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)
4I13 2013-11-27 1.6 Nanobody ca1697 binding to the DHFR.folate binary complex
1RA2 1996-12-23 1.6 DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)
5UIH 2017-11-22 1.65 structure of DHFR with bound phenformin and NADP
4DFR 1982-10-21 1.7 CRYSTAL STRUCTURES OF ESCHERICHIA COLI AND LACTOBACILLUS CASEI DIHYDROFOLATE REDUCTASE REFINED AT 1.7 ANGSTROMS RESOLUTION. I. GENERAL FEATURES AND BINDING OF METHOTREXATE
5EAJ 2016-09-21 1.7 Crystal structure of DHFR in 0% Isopropanol
4X5F 2016-01-13 1.7 ecDHFR complexed with folate and NADP+ at 0.1 MPa
4EJ1 2013-04-24 1.75 Binding of Nb113 camelid antibody fragment with the binary DHFR:folate complex
3OCH 2010-12-08 1.79 Chemically Self-assembled Antibody Nanorings (CSANs): Design and Characterization of an Anti-CD3 IgM Biomimetic
3QYL 2012-01-18 1.79 Sensitivity of receptor internal motions to ligand binding affinity and kinetic off-rate
5UJX 2017-12-27 1.8 Crystal structure of DHFR in 20% Isopropanol
1RF7 1997-03-12 1.8 STRUCTURE OF DIHYDROFOLATE REDUCTASE COMPLEXED WITH DIHYDROFOLATE
3QL3 2011-04-27 1.8 Re-refined coordinates for PDB entry 1RX2
5Z6F 2018-09-19 1.8 High-pressure Crystal Structure Analysis of DHFR(0.1 MPa)
1RA8 1997-03-12 1.8 DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE AND 2-MONOPHOSPHOADENOSINE 5'-DIPHOSPHORIBOSE
1DHJ 1994-01-31 1.8 LONG-RANGE STRUCTURAL EFFECTS IN A SECOND-SITE REVERTANT OF A MUTANT DIHYDROFOLATE REDUCTASE
5Z6K 2018-09-19 1.8 High-pressure Crystal Structure Analysis of M20 loop closed DHFR at 400 MPa
4X5I 2016-01-13 1.8 ecDHFR complexed with folate and NADP+ at 660 MPa
5E8Q 2016-09-21 1.8 Crystal structure of DHFR in 20% Isopropanol
1RX2 1997-01-11 1.8 DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH WITH FOLATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)
1RA3 1996-12-23 1.8 DIHYDROFOLATE REDUCTASE COMPLEXED WITH METHOTREXATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)
5Z6J 2018-09-19 1.8 High-pressure Crystal Structure Analysis of M20 loop closed DHFR at 220 MPa
4X5J 2016-01-13 1.85 ecDHFR complexed with folate and NADP+ at 750 MPa
1DYJ 1995-06-03 1.85 ISOMORPHOUS CRYSTAL STRUCTURES OF ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE, 5-DEAZAFOLATE AND 5,10-DIDEAZATETRAHYDROFOLATE: MECHANISTIC IMPLICATIONS
4GH8 2013-06-05 1.85 Crystal structure of a 'humanized' E. coli dihydrofolate reductase
4I1N 2013-11-27 1.89 R104A-ca1697 nanobody binding to the binary DHFR.folate complex
1DHI 1994-01-31 1.9 LONG-RANGE STRUCTURAL EFFECTS IN A SECOND-SITE REVERTANT OF A MUTANT DIHYDROFOLATE REDUCTASE
1DYH 1995-06-03 1.9 ISOMORPHOUS CRYSTAL STRUCTURES OF ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE, 5-DEAZAFOLATE AND 5,10-DIDEAZATETRAHYDROFOLATE: MECHANISTIC IMPLICATIONS
1DRA 1994-01-31 1.9 CRYSTAL STRUCTURE OF UNLIGANDED ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE. LIGAND-INDUCED CONFORMATIONAL CHANGES AND COOPERATIVITY IN BINDING
5UIP 2017-11-22 1.9 structure of DHFR with bound DAP, p-ABG and NADP
2D0K 2006-02-28 1.9 Methionine-free mutant of Escherichia coli dihydrofolate reductase
3DRC 1994-01-31 1.9 INVESTIGATION OF THE FUNCTIONAL ROLE OF TRYPTOPHAN-22 IN ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE BY SITE-DIRECTED MUTAGENESIS
2DRC 1994-01-31 1.9 INVESTIGATION OF THE FUNCTIONAL ROLE OF TRYPTOPHAN-22 IN ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE BY SITE-DIRECTED MUTAGENESIS
1JOM 1996-11-08 1.9 THE CRYSTAL STRUCTURE OF THE BINARY COMPLEX BETWEEN FOLINIC ACID (LEUCOVORIN) AND E. COLI DIHYDROFOLATE REDUCTASE
4X5G 2016-01-13 1.9 ecDHFR complexed with folate and NADP+ at 270 MPa
1DYI 1995-06-03 1.9 ISOMORPHOUS CRYSTAL STRUCTURES OF ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE, 5-DEAZAFOLATE AND 5,10-DIDEAZATETRAHYDROFOLATE: MECHANISTIC IMPLICATIONS
5Z6L 2018-09-19 1.9 High-pressure Crystal Structure Analysis of M20 loop closed DHFR at 650 MPa
4X5H 2016-01-13 1.9 ecDHFR complexed with folate and NADP+ at 500 MPa
1RA1 1996-12-23 1.9 DIHYDROFOLATE REDUCTASE COMPLEXED WITH NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (REDUCED FORM)
1RC4 1997-03-12 1.9 DIHYDROFOLATE REDUCTASE COMPLEXED WITH 5,10-DIDEAZATETRAHYDROFOLATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)
1RX9 1997-04-01 1.9 DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)
5UIO 2017-11-22 1.93 structure of DHFR with bound DAP, p-ABG and NADP
3K74 2010-10-20 1.95 Disruption of protein dynamics by an allosteric effector antibody
1DRB 1994-01-31 1.96 CRYSTAL STRUCTURE OF UNLIGANDED ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE. LIGAND-INDUCED CONFORMATIONAL CHANGES AND COOPERATIVITY IN BINDING
1JOL 1996-11-08 1.96 THE CRYSTAL STRUCTURE OF THE BINARY COMPLEX BETWEEN FOLINIC ACID (LEUCOVORIN) AND E. COLI DIHYDROFOLATE REDUCTASE
1RX1 1997-01-11 2.0 DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (REDUCED FORM)
1RG7 1997-03-12 2.0 DIHYDROFOLATE REDUCTASE COMPLEXED WITH METHOTREXATE
1RX6 1997-01-11 2.0 DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH 5,10-DIDEAZATETRAHYDROFOLATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (REDUCED FORM)
3KFY 2010-12-08 2.08 Dynamic switching and partial occupancies of a small molecule inhibitor complex of DHFR
3R33 2012-01-25 2.09 Evidence for dynamic motion in proteins as a mechanism for ligand dissociation
3QYO 2012-01-18 2.09 Sensitivity of receptor internal motions to ligand binding affinity and kinetic off-rate
1RB2 1997-03-12 2.1 DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)
2ANQ 2006-07-25 2.13 Crystal Structure of E.coli DHFR in complex with NADPH and the inhibitor compound 10a.
6CQA 2019-01-09 2.2 E. coli DHFR complex with inhibitor AMPQD
1RX4 1997-01-11 2.2 DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH 5,10-DIDEAZATETRAHYDROFOLATE AND 2'-MONOPHOSPHOADENOSINE 5'-DIPHOSPHORIBOSE
5Z6M 2018-09-19 2.2 High-pressure Crystal Structure Analysis of M20 loop closed DHFR at 800 MPa
2INQ 2007-02-06 2.2 Neutron Crystal Structure of Escherichia coli Dihydrofolate Reductase Bound to the Anti-cancer drug, Methotrexate
1RX3 1997-01-11 2.2 DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH METHOTREXATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (REDUCED FORM)
4EIZ 2013-04-24 2.2 Structure of Nb113 bound to apoDHFR
1DDS 1995-10-15 2.2 MOLECULE: DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH METHOTREXATE
1RX5 1997-01-11 2.3 DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH 5,10-DIDEAZATETRAHYDROFOLATE
1RB3 1997-03-12 2.3 DIHYDROFOLATE REDUCTASE COMPLEXED WITH METHOTREXATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)
5DFR 1990-07-15 2.3 CRYSTAL STRUCTURE OF UNLIGANDED ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE. LIGAND-INDUCED CONFORMATIONAL CHANGES AND COOPERATIVITY IN BINDING
1RX7 1997-03-12 2.3 STRUCTURE OF DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE
1DRH 1995-02-07 2.3 ISOMORPHOUS CRYSTAL STRUCTURES OF ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE, 5-DEAZAFOLATE AND 5,10-DIDEAZATETRAHYDROFOLATE: MECHANISTIC IMPLICATIONS
1RH3 1997-03-12 2.4 DIHYDROFOLATE REDUCTASE COMPLEXED WITH METHOTREXATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (REDUCED FORM)
6DFR 1990-07-15 2.4 CRYSTAL STRUCTURES OF ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE. THE NADP+ HOLOENZYME AND THE FOLATE(DOT)NADP+ TERNARY COMPLEX. SUBSTRATE BINDING AND A MODEL FOR THE TRANSITION STATE
1DDR 1995-10-15 2.45 MOLECULE: DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH METHOTREXATE AND UREA
1TDR 1995-07-10 2.5 EXPRESSION, CHARACTERIZATION, AND CRYSTALLOGRAPHIC ANALYSIS OF TELLUROMETHIONYL DIHYDROFOLATE REDUCTASE
7DFR 1990-07-15 2.5 CRYSTAL STRUCTURES OF ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE. THE NADP+ HOLOENZYME AND THE FOLATE(DOT)NADP+ TERNARY COMPLEX. SUBSTRATE BINDING AND A MODEL FOR THE TRANSITION STATE
4EIG 2013-04-24 2.5 CA1698 camel antibody fragment in complex with DHFR
1RD7 1996-12-23 2.6 DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE
1RE7 1996-12-23 2.6 DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE
1DRE 1997-03-12 2.6 DIHYDROFOLATE REDUCTASE COMPLEXED WITH METHOTREXATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)
2ANO 2006-07-25 2.68 Crystal structure of E.coli dihydrofolate reductase in complex with NADPH and the inhibitor MS-SH08-17
1RX8 1997-03-12 2.8 DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE AND 2'-MONOPHOSPHOADENOSINE 5'-DIPHOSPHORIBOSE
4FHB 2013-04-24 2.8 Enhancing DHFR catalysis by binding of an allosteric regulator nanobody (Nb179)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
91.8 Dihydrofolate reductase P31074 DYR_KLEAE
96.2 Dihydrofolate reductase P31073 DYR_CITFR
100.0 Dihydrofolate reductase P0ABQ6 DYR_SHIFL
100.0 Dihydrofolate reductase P0ABQ4 DYR_ECOLI
100.0 Dihydrofolate reductase P0ABQ5 DYR_ECOL6