1GOA

COOPERATIVE STABILIZATION OF ESCHERICHIA COLI RIBONUCLEASE HI BY INSERTION OF GLY-80B AND GLY-77-> ALA SUBSTITUTION

Summary for 1GOA

• Entry DOI: 10.2210/pdb1goa/pdb
• Related: 2RN2
• Descriptor: RIBONUCLEASE H (2 entities in total)
• Functional Keywords: hydrolase(endoribonuclease)
• Biological source: Escherichia coli
• Cellular location: Cytoplasm : P0A7Y4
• Total number of polymer chains: 1
• Total formula weight: 17680.05

Authors

Ishikawa, K.,Kimura, S.,Nakamura, H.,Morikawa, K.,Kanaya, S. (deposition date: 1993-05-10, release date: 1994-01-31, Last modification date: 2024-02-07)

Primary citation

Ishikawa, K.,Nakamura, H.,Morikawa, K.,Kimura, S.,Kanaya, S.
Cooperative stabilization of Escherichia coli ribonuclease HI by insertion of Gly-80b and Gly-77-->Ala substitution.
Biochemistry, 32:7136-7142, 1993

PubMed 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)

PubMed: 8393706
DOI: 10.1021/bi00079a010

Experimental method

X-RAY DIFFRACTION (1.9 Å)