Cooperative stabilization of Escherichia coli ribonuclease HI by insertion of Gly-80b and Gly-77-->Ala substitution.


Abstract

The insertion of a Gly residue (designated as Gly-80b) between the C-cap of the alpha II-helix (Gln-80) and the N-cap of the alpha III-helix (Trp-81) in Escherichia coli ribonuclease HI enhances the protein stability by 0.4 kcal/mol in delta G (Kimura, S., Nakamura, H., Hashimoto, T., Oobatake, M., & Kanaya, S. (1992) J. Biol. Chem. 267, 21535-21542). Another mutation within the alpha II-helix, Gly-77-->Ala, reduces the stability by 0.9 kcal/mol. Simultaneous introduction of these mutations enhances the stability by 0.8 kcal/mol, indicating that the effects of these mutations are cooperative and not simply independent. We determined the crystal structures of these three mutant proteins (G80b-, A77-, and A77/G80b-RNase H) to investigate this cooperative mechanism of the protein stabilization. The structures revealed that the inserted Gly-80b assumes a left-handed helical conformation in both the G80b- and the A77/G80b-RNase H. This inserted glycine residue allows the formation of a "paperclip", which is a common motif at the C-termini of alpha-helices. Accompanying the formation of the paperclip motif, two intrahelical hydrogen bonds are formed between the backbone atoms (O78-N80b and O80b-N84). The stabilization caused by the insertion of Gly-80b can be ascribed to the formation of these hydrogen bonds. The Gly-77-->Ala substitution destabilizes the protein due to the deformed packing interactions in the hydrophobic core around Ala-77 and the stress in the wedged indole ring of Trp-81. These effects are alleviated by the insertion of Gly-80b, which relaxes the backbone structure.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 732, 733, 13371 Extra Details: Escherichia coli ribonuclease HI; cooperative stabilization;,crystal structures; alpha-helices; stability

Submission Details

ID: nVY4JBCW4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:15 p.m.

Version: 1

Publication Details
Ishikawa K;Nakamura H;Morikawa K;Kimura S;Kanaya S,Biochemistry (1993) Cooperative stabilization of Escherichia coli ribonuclease HI by insertion of Gly-80b and Gly-77-->Ala substitution. PMID:8393706
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
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
3QIO 2011-04-20 1.4 Crystal Structure of HIV-1 RNase H with engineered E. coli loop and N-hydroxy quinazolinedione inhibitor
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
1RBR 1994-01-31 1.8 STRUCTURAL STUDY OF MUTANTS OF ESCHERICHIA COLI RIBONUCLEASE HI WITH ENHANCED THERMOSTABILITY
1RBU 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
1KVA 1997-03-12 1.8 E. COLI RIBONUCLEASE HI D134A MUTANT
1RBT 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
1LAV 1993-10-31 1.8 STABILIZATION OF ESCHERICHIA COLI RIBONUCLEASE HI BY CAVITY-FILLING MUTATIONS WITHIN A HYDROPHOBIC CORE
1LAW 1993-10-31 1.8 STABILIZATION OF ESCHERICHIA COLI RIBONUCLEASE HI BY CAVITY-FILLING MUTATIONS WITHIN A HYDROPHOBIC CORE
1GOA 1994-01-31 1.9 COOPERATIVE STABILIZATION OF ESCHERICHIA COLI RIBONUCLEASE HI BY INSERTION OF GLY-80B AND GLY-77-> ALA SUBSTITUTION
1G15 2001-03-14 1.9 CO-CRYSTAL OF E. COLI RNASE HI WITH TWO MN2+ IONS BOUND IN THE THE ACTIVE SITE
3AA2 2010-10-06 1.9 A52I E. coli RNase HI
1KVB 1997-03-12 1.9 E. COLI RIBONUCLEASE HI D134H MUTANT
1RDB 1993-10-31 1.9 CRYSTAL STRUCTURES OF RIBONUCLEASE HI ACTIVE SITE MUTANTS FROM ESCHERICHIA COLI
1KVC 1997-03-12 1.9 E. COLI RIBONUCLEASE HI D134N MUTANT
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
1GOB 1994-01-31 2.0 COOPERATIVE STABILIZATION OF ESCHERICHIA COLI RIBONUCLEASE HI BY INSERTION OF GLY-80B AND GLY-77-> ALA SUBSTITUTION
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
3AA3 2010-10-06 2.2 A52L E. coli RNase HI
1WSG 2005-02-08 2.2 Co-crystal structure of E.coli RNase HI active site mutant (E48A/D134N*) with Mn2+
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