The effect of salts on the activity and stability of Escherichia coli and Haloferax volcanii dihydrofolate reductases.


Abstract

The extremely halophilic Archae require near-saturating concentrations of salt in the external environment and in their cytoplasm, potassium being the predominant intracellular cation. The proteins of these organisms have evolved to function in concentrations of salt that inactivate or precipitate homologous proteins from non-halophilic species. It has been proposed that haloadaptation is primarily due to clustering of acidic residues on the surface of the protein, and that these clusters bind networks of hydrated ions. The dihydrofolate reductases from Escherichia coli (ecDHFR) and two DHFR isozymes from Haloferax volcanii (hvDHFR1 and hvDHFR2) have been used as a model system to compare the effect of salts on a mesophilic and halophilic enzyme. The KCl-dependence of the activity and substrate affinity was investigated. ecDHFR is largely inactivated above 1M KCl, with no major effect on substrate affinity. hvDHFR1 and hvDHFR2 unfold at KCl concentrations below approximately 0.5M. Above approximately 1M, the KCl dependence of the hvDHFR activities can be attributed to the effect of salt on substrate affinity. The abilities of NaCl, KCl, and CsCl to enhance the stability to urea denaturation were determined, and similar efficacies of stabilization were observed for all three DHFR variants. The DeltaG degrees (H(2)O) values increased linearly with increasing KCl and CsCl concentrations. The increase of DeltaG degrees (H(2)O) as a function of the smallest cation, NaCl, is slightly curved, suggesting a minor stabilization from cation binding or screening of electrostatic repulsion. At their respective physiological ionic strengths, the DHFR variants exhibit similar stabilities. Salts stabilize ecDHFR and the hvDHFRs by a common mechanism, not a halophile-specific mechanism, such as the binding of hydrated salt networks. The primary mode of salt stabilization of the mesophilic and halophilic DHFRs appears to be through preferential hydration and the Hofmeister effect of salt on the activity and entropy of the aqueous solvent. In support of this conclusion, all three DHFRs are similarly stabilized by the non-ionic cosolute, sucrose. Study holds ProTherm entries: 17802, 17803, 17804, 17805, 17806, 17807, 17808, 17809, 17810, 17811, 17812, 17813 Extra Details: protein folding; enzymology; circular dichroism; fluorescence; halophilic enzymes

Submission Details

ID: L246pVW9

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:50 p.m.

Version: 1

Publication Details
Wright DB;Banks DD;Lohman JR;Hilsenbeck JL;Gloss LM,J. Mol. Biol. (2002) The effect of salts on the activity and stability of Escherichia coli and Haloferax volcanii dihydrofolate reductases. PMID:12381324
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
5UII 2018-01-31 1.35 structure of DHFR with bound buformin and NADP
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
6MTH 2019-05-15 1.35 E. coli DHFR complex modeled with three ligand states
4KJK 2013-08-21 1.35 Room Temperature WT DHFR
4NX6 2014-01-15 1.35 single room temperature model of DHFR
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
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
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
4EJ1 2013-04-24 1.75 Binding of Nb113 camelid antibody fragment with the binary DHFR:folate complex
3QYL 2012-01-18 1.79 Sensitivity of receptor internal motions to ligand binding affinity and kinetic off-rate
3OCH 2010-12-08 1.79 Chemically Self-assembled Antibody Nanorings (CSANs): Design and Characterization of an Anti-CD3 IgM Biomimetic
5UJX 2017-12-27 1.8 Crystal structure of DHFR in 20% Isopropanol
5Z6K 2018-09-19 1.8 High-pressure Crystal Structure Analysis of M20 loop closed DHFR at 400 MPa
1RA3 1996-12-23 1.8 DIHYDROFOLATE REDUCTASE COMPLEXED WITH METHOTREXATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)
4X5I 2016-01-13 1.8 ecDHFR complexed with folate and NADP+ at 660 MPa
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)
5E8Q 2016-09-21 1.8 Crystal structure of DHFR in 20% Isopropanol
1RA8 1997-03-12 1.8 DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE AND 2-MONOPHOSPHOADENOSINE 5'-DIPHOSPHORIBOSE
1RF7 1997-03-12 1.8 STRUCTURE OF DIHYDROFOLATE REDUCTASE COMPLEXED WITH DIHYDROFOLATE
1DHJ 1994-01-31 1.8 LONG-RANGE STRUCTURAL EFFECTS IN A SECOND-SITE REVERTANT OF A MUTANT DIHYDROFOLATE REDUCTASE
3QL3 2011-04-27 1.8 Re-refined coordinates for PDB entry 1RX2
5Z6J 2018-09-19 1.8 High-pressure Crystal Structure Analysis of M20 loop closed DHFR at 220 MPa
5Z6F 2018-09-19 1.8 High-pressure Crystal Structure Analysis of DHFR(0.1 MPa)
4X5J 2016-01-13 1.85 ecDHFR complexed with folate and NADP+ at 750 MPa
4GH8 2013-06-05 1.85 Crystal structure of a 'humanized' E. coli dihydrofolate reductase
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
4I1N 2013-11-27 1.89 R104A-ca1697 nanobody binding to the binary DHFR.folate complex
1JOM 1996-11-08 1.9 THE CRYSTAL STRUCTURE OF THE BINARY COMPLEX BETWEEN FOLINIC ACID (LEUCOVORIN) AND E. 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
1RA1 1996-12-23 1.9 DIHYDROFOLATE REDUCTASE COMPLEXED WITH NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (REDUCED FORM)
5UIP 2017-11-22 1.9 structure of DHFR with bound DAP, p-ABG and NADP
1DHI 1994-01-31 1.9 LONG-RANGE STRUCTURAL EFFECTS IN A SECOND-SITE REVERTANT OF A MUTANT DIHYDROFOLATE REDUCTASE
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
4X5G 2016-01-13 1.9 ecDHFR complexed with folate and NADP+ at 270 MPa
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
2D0K 2006-02-28 1.9 Methionine-free mutant of Escherichia coli dihydrofolate reductase
1DRA 1994-01-31 1.9 CRYSTAL STRUCTURE OF UNLIGANDED ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE. LIGAND-INDUCED CONFORMATIONAL CHANGES AND COOPERATIVITY IN BINDING
1RC4 1997-03-12 1.9 DIHYDROFOLATE REDUCTASE COMPLEXED WITH 5,10-DIDEAZATETRAHYDROFOLATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)
2DRC 1994-01-31 1.9 INVESTIGATION OF THE FUNCTIONAL ROLE OF TRYPTOPHAN-22 IN ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE BY SITE-DIRECTED MUTAGENESIS
1RX9 1997-04-01 1.9 DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)
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
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
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)
1RG7 1997-03-12 2.0 DIHYDROFOLATE REDUCTASE COMPLEXED WITH METHOTREXATE
1RX1 1997-01-11 2.0 DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH 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.
1RX3 1997-01-11 2.2 DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH METHOTREXATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (REDUCED FORM)
1RX4 1997-01-11 2.2 DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH 5,10-DIDEAZATETRAHYDROFOLATE AND 2'-MONOPHOSPHOADENOSINE 5'-DIPHOSPHORIBOSE
6CQA 2019-01-09 2.2 E. coli DHFR complex with inhibitor AMPQD
5Z6M 2018-09-19 2.2 High-pressure Crystal Structure Analysis of M20 loop closed DHFR at 800 MPa
1DDS 1995-10-15 2.2 MOLECULE: DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH METHOTREXATE
4EIZ 2013-04-24 2.2 Structure of Nb113 bound to apoDHFR
2INQ 2007-02-06 2.2 Neutron Crystal Structure of Escherichia coli Dihydrofolate Reductase Bound to the Anti-cancer drug, Methotrexate
1RX5 1997-01-11 2.3 DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH 5,10-DIDEAZATETRAHYDROFOLATE
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
5DFR 1990-07-15 2.3 CRYSTAL STRUCTURE OF UNLIGANDED ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE. LIGAND-INDUCED CONFORMATIONAL CHANGES AND COOPERATIVITY IN BINDING
1RB3 1997-03-12 2.3 DIHYDROFOLATE REDUCTASE COMPLEXED WITH METHOTREXATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED 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
1RH3 1997-03-12 2.4 DIHYDROFOLATE REDUCTASE COMPLEXED WITH METHOTREXATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (REDUCED FORM)
1DDR 1995-10-15 2.45 MOLECULE: DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH METHOTREXATE AND UREA
4EIG 2013-04-24 2.5 CA1698 camel antibody fragment in complex with DHFR
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
1RD7 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)
1RE7 1996-12-23 2.6 DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE
2ANO 2006-07-25 2.68 Crystal structure of E.coli dihydrofolate reductase in complex with NADPH and the inhibitor MS-SH08-17
4FHB 2013-04-24 2.8 Enhancing DHFR catalysis by binding of an allosteric regulator nanobody (Nb179)
1RX8 1997-03-12 2.8 DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE AND 2'-MONOPHOSPHOADENOSINE 5'-DIPHOSPHORIBOSE

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