Thermal stability of Escherichia coli ribonuclease HI and its active site mutants in the presence and absence of the Mg2+ ion. Proposal of a novel catalytic role for Glu48.


Abstract

Escherichia coli ribonuclease HI, which requires divalent cations (Mg2+ or Mn2+) for activity, was thermostabilized by 2.6-3.0 kcal/mol in the presence of the Mg2+, Mn2+, or Ca2+ ion, probably because the negative charge repulsion around the active site was canceled upon the binding of these metal ions. The dissociation constants were determined to be 0.71 mM for Mg2+, 0.035 mM for Mn2+, and 0.16 mM for Ca2+. Likewise, various active site mutants at Asp10, Glu48, Asp70, or Asp134 were thermostabilized by 0.4-3.0 kcal/mol in the presence of the Mg2+ ion, suggesting that this ion binds to these mutant proteins as well. The dissociation constants of Mg2+ were determined to be 9.8 mM for D10N, 1.1 mM for E48Q, 18.8 mM for D70N, and 1.8 mM for D134N. Thus, the mutation of Asp10 or Asp70 to Asn considerably impairs the Mg2+ binding, whereas the mutation of Glu48 to Gln or Asp134 to Asn does not. Comparison of the thermal stability of the mutant proteins with that of the wild-type protein in the absence of the Mg2+ ion suggests that the negative charge repulsion between Asp10 and Asp70 is responsible for the binding of the metal cofactor. Glu48 may be required to anchor a water molecule, which functions as a general acid. Study holds ProTherm entries: 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 13216, 13217, 13218, 13219, 13220, 13221, 13222, 13223, 13224, 13225, 13226, 13227, 13228, 13229, 13230, 13231, 13232, 13233, 13234, 13235, 13236, 13237, 13238, 13239, 13240, 13241 Extra Details: Escherichia coli ribonuclease HI; thermal stability; catalytic role;,dissociation constant

Submission Details

ID: 6yGKkEH93

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:14 p.m.

Version: 1

Publication Details
Kanaya S;Oobatake M;Liu Y,J. Biol. Chem. (1996) Thermal stability of Escherichia coli ribonuclease HI and its active site mutants in the presence and absence of the Mg2+ ion. Proposal of a novel catalytic role for Glu48. PMID:8955106
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
1RCH 1997-02-12 SOLUTION NMR STRUCTURE OF RIBONUCLEASE HI FROM ESCHERICHIA COLI, 8 STRUCTURES
1JL1 2002-02-27 1.3 D10A E. coli ribonuclease HI
3QIO 2011-04-20 1.4 Crystal Structure of HIV-1 RNase H with engineered E. coli loop and N-hydroxy quinazolinedione inhibitor
2YV0 2008-03-18 1.4 Structural and Thermodynamic Analyses of E. coli ribonuclease HI Variant with Quintuple Thermostabilizing Mutations
1F21 2000-12-06 1.4 DIVALENT METAL COFACTOR BINDING IN THE KINETIC FOLDING TRAJECTORY OF E. COLI RIBONUCLEASE HI
2RN2 1993-10-31 1.48 STRUCTURAL DETAILS OF RIBONUCLEASE H FROM ESCHERICHIA COLI AS REFINED TO AN ATOMIC RESOLUTION
1JXB 2002-03-06 1.6 I53A, a point mutant of the cysteine-free variant of E. coli Rnase HI
3HYF 2009-10-20 1.7 Crystal structure of HIV-1 RNase H p15 with engineered E. coli loop and active site inhibitor
3QIN 2011-04-20 1.7 Crystal Structure of HIV-1 RNase H p15 with engineered E. coli loop and pyrimidinol carboxylic acid inhibitor
1JL2 2002-01-18 1.76 Crystal structure of TCEO RNase H-a chimera combining the folding core from T. thermophilus RNase H and the remaining region of E. coli RNase H
3AA4 2010-10-06 1.79 A52V E.coli RNase HI
1RBU 1994-01-31 1.8 STRUCTURAL STUDY OF MUTANTS OF ESCHERICHIA COLI RIBONUCLEASE HI WITH ENHANCED THERMOSTABILITY
1RBS 1994-01-31 1.8 STRUCTURAL STUDY OF MUTANTS OF ESCHERICHIA COLI RIBONUCLEASE HI WITH ENHANCED THERMOSTABILITY
1LAW 1993-10-31 1.8 STABILIZATION OF ESCHERICHIA COLI RIBONUCLEASE HI BY CAVITY-FILLING MUTATIONS WITHIN A HYDROPHOBIC CORE
1RBT 1994-01-31 1.8 STRUCTURAL STUDY OF MUTANTS OF ESCHERICHIA COLI RIBONUCLEASE HI WITH ENHANCED THERMOSTABILITY
1RBV 1994-01-31 1.8 STRUCTURAL STUDY OF MUTANTS OF ESCHERICHIA COLI RIBONUCLEASE HI WITH ENHANCED THERMOSTABILITY
1RBR 1994-01-31 1.8 STRUCTURAL STUDY OF MUTANTS OF ESCHERICHIA COLI RIBONUCLEASE HI WITH ENHANCED THERMOSTABILITY
1LAV 1993-10-31 1.8 STABILIZATION OF ESCHERICHIA COLI RIBONUCLEASE HI BY CAVITY-FILLING MUTATIONS WITHIN A HYDROPHOBIC CORE
1KVA 1997-03-12 1.8 E. COLI RIBONUCLEASE HI D134A MUTANT
3AA2 2010-10-06 1.9 A52I E. coli RNase HI
1GOA 1994-01-31 1.9 COOPERATIVE STABILIZATION OF ESCHERICHIA COLI RIBONUCLEASE HI BY INSERTION OF GLY-80B AND GLY-77-> ALA SUBSTITUTION
1RDB 1993-10-31 1.9 CRYSTAL STRUCTURES OF RIBONUCLEASE HI ACTIVE SITE MUTANTS FROM ESCHERICHIA COLI
1KVB 1997-03-12 1.9 E. COLI RIBONUCLEASE HI D134H MUTANT
1KVC 1997-03-12 1.9 E. COLI RIBONUCLEASE HI D134N MUTANT
1G15 2001-03-14 1.9 CO-CRYSTAL OF E. COLI RNASE HI WITH TWO MN2+ IONS BOUND IN THE THE ACTIVE SITE
1GOB 1994-01-31 2.0 COOPERATIVE STABILIZATION OF ESCHERICHIA COLI RIBONUCLEASE HI BY INSERTION OF GLY-80B AND GLY-77-> ALA SUBSTITUTION
4Z0U 2015-04-29 2.0 RNase HI/SSB-Ct complex
2Z1I 2007-11-13 2.0 Crystal structure of E.coli RNase HI surface charged mutant(Q4R/T40E/Q72H/Q76K/Q80E/T92K/Q105K/Q113R/Q115K)
1RNH 1991-10-15 2.0 STRUCTURE OF RIBONUCLEASE H PHASED AT 2 ANGSTROMS RESOLUTION BY MAD ANALYSIS OF THE SELENOMETHIONYL PROTEIN
1GOC 1994-01-31 2.0 COOPERATIVE STABILIZATION OF ESCHERICHIA COLI RIBONUCLEASE HI BY INSERTION OF GLY-80B AND GLY-77-> ALA SUBSTITUTION
3AA5 2010-10-06 2.1 A52F E.coli RNase HI
2Z1G 2007-11-13 2.1 Crystal structure of E.coli RNase HI surface charged mutant(Q4R/T40E/Q72H/Q76K/Q80E/T92K/Q105K)
1RDA 1993-10-31 2.15 CRYSTAL STRUCTURES OF RIBONUCLEASE HI ACTIVE SITE MUTANTS FROM ESCHERICHIA COLI
1WSG 2005-02-08 2.2 Co-crystal structure of E.coli RNase HI active site mutant (E48A/D134N*) with Mn2+
3AA3 2010-10-06 2.2 A52L E. coli RNase HI
1WSF 2005-02-08 2.3 Co-crystal structure of E.coli RNase HI active site mutant (D134A*) with Mn2+
1WSE 2005-02-08 2.3 Co-crystal structure of E.coli RNase HI active site mutant (E48A*) with Mn2+
1RDC 1993-10-31 2.3 CRYSTAL STRUCTURES OF RIBONUCLEASE HI ACTIVE SITE MUTANTS FROM ESCHERICHIA COLI
2Z1J 2007-11-13 2.38 Crystal structure of E.coli RNase HI surface charged mutant(Q4R/T40E/Q72H/Q76K/Q80E/T92K/Q105K/Q113R/Q115K/N143K/T145K)
2Z1H 2007-11-13 2.6 Crystal structure of E.coli RNase HI surface charged mutant(Q4R/T92K/Q105K/Q113R/Q115K/N143K/T145K)
1RDD 1993-10-31 2.8 CRYSTAL STRUCTURE OF ESCHERICHIA COLI RNASE HI IN COMPLEX WITH MG2+ AT 2.8 ANGSTROMS RESOLUTION: PROOF FOR A SINGLE MG2+ SITE

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
90.9 Ribonuclease HI A6T512 RNH_KLEP7
90.9 Ribonuclease HI B5Y1G2 RNH_KLEP3
92.9 Ribonuclease HI C0Q6N2 RNH_SALPC
93.5 Ribonuclease HI P0A2B9 RNH_SALTY
93.5 Ribonuclease HI P0A2C0 RNH_SALTI
93.5 Ribonuclease HI B4TYH0 RNH_SALSV
93.5 Ribonuclease HI B5BDW5 RNH_SALPK
93.5 Ribonuclease HI A9MZ19 RNH_SALPB
93.5 Ribonuclease HI Q5PFD8 RNH_SALPA
93.5 Ribonuclease HI B4SV39 RNH_SALNS
93.5 Ribonuclease HI B4TK85 RNH_SALHS
93.5 Ribonuclease HI B5R5L3 RNH_SALG2
93.5 Ribonuclease HI B5R449 RNH_SALEP
93.5 Ribonuclease HI B5FJ58 RNH_SALDC
93.5 Ribonuclease HI Q57SZ6 RNH_SALCH
93.5 Ribonuclease HI A9MPF1 RNH_SALAR
93.5 Ribonuclease HI B5F8X2 RNH_SALA4
93.5 Ribonuclease HI A8AKR0 RNH_CITK8
99.4 Ribonuclease HI Q0TLC3 RNH_ECOL5
99.4 Ribonuclease HI A7ZWF6 RNH_ECOHS
99.4 Ribonuclease HI B7LHC0 RNH_ECO55
99.4 Ribonuclease HI B7UJB0 RNH_ECO27
100.0 Ribonuclease HI Q3Z5E9 RNH_SHISS
100.0 Ribonuclease HI P0A7Y7 RNH_SHIFL
100.0 Ribonuclease HI Q32JP9 RNH_SHIDS
100.0 Ribonuclease HI Q325T2 RNH_SHIBS
100.0 Ribonuclease HI B2U352 RNH_SHIB3
100.0 Ribonuclease HI B7LW89 RNH_ESCF3
100.0 Ribonuclease HI B1LHM3 RNH_ECOSM
100.0 Ribonuclease HI B6HZS7 RNH_ECOSE
100.0 Ribonuclease HI B7N876 RNH_ECOLU
100.0 Ribonuclease HI P0A7Y4 RNH_ECOLI
100.0 Ribonuclease HI B1IPU4 RNH_ECOLC
100.0 Ribonuclease HI P0A7Y5 RNH_ECOL6
100.0 Ribonuclease HI B1XD78 RNH_ECODH
100.0 Ribonuclease HI C4ZRV1 RNH_ECOBW
100.0 Ribonuclease HI B7M213 RNH_ECO8A
100.0 Ribonuclease HI B7MQ23 RNH_ECO81
100.0 Ribonuclease HI B7NKW4 RNH_ECO7I
100.0 Ribonuclease HI B5Z0I8 RNH_ECO5E
100.0 Ribonuclease HI P0A7Y6 RNH_ECO57
100.0 Ribonuclease HI B7MBJ0 RNH_ECO45
100.0 Ribonuclease HI A7ZHV1 RNH_ECO24