PDBsum entry 1box

Hydrolase

PDB id: 1box

Contents

Protein chain

95 a.a. *

Waters ×95

* Residue conservation analysis

PDB id:

1box

Name:

Hydrolase

Title:

N39s mutant of rnase sa from streptomyces aureofaciens

Structure:

Guanyl-specific ribonuclease sa. Chain: a. Engineered: yes. Mutation: yes

Source:

Streptomyces aureofaciens. Organism_taxid: 1894. Strain: bmk. Gene: u39467. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.60Å R-factor: 0.176 R-free: 0.218

Authors:

E.J.Hebert,A.Giletto,J.Sevcik,L.Urbanikova,K.S.Wilson,Z.Dauter, C.N.Pace

Key ref:

E.J.Hebert et al. (1998). Contribution of a conserved asparagine to the conformational stability of ribonucleases Sa, Ba, and T1. Biochemistry, 37, 16192-16200. PubMed id: 9819211 DOI: 10.1021/bi9815243

Date:

07-Aug-98 Release date: 29-Dec-99

Protein chain

P05798  (RNSA_KITAU) -  Guanyl-specific ribonuclease Sa from Kitasatospora aureofaciens

Seq: 96 a.a.

Struc: 95 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.4.6.1.24 - ribonuclease T1.
• Reaction: [RNA] containing guanosine + H2O = an [RNA fragment]-3'-guanosine- 3'-phosphate + a 5'-hydroxy-ribonucleotide-3'-[RNA fragment]

DOI no: 10.1021/bi9815243

Biochemistry 37:16192-16200 (1998)

PubMed id: 9819211

Contribution of a conserved asparagine to the conformational stability of ribonucleases Sa, Ba, and T1.

E.J.Hebert, A.Giletto, J.Sevcik, L.Urbanikova, K.S.Wilson, Z.Dauter, C.N.Pace.

ABSTRACT

The contribution of hydrogen bonding by peptide groups to the conformational stability of globular proteins was studied. One of the conserved residues in the microbial ribonuclease (RNase) family is an asparagine at position 39 in RNase Sa, 44 in RNase T1, and 58 in RNase Ba (barnase). The amide group of this asparagine is buried and forms two similar intramolecular hydrogen bonds with a neighboring peptide group to anchor a loop on the surface of all three proteins. Thus, it is a good model for the hydrogen bonding of peptide groups. When the conserved asparagine is replaced with alanine, the decrease in the stability of the mutant proteins is 2.2 (Sa), 1.8 (T1), and 2.7 (Ba) kcal/mol. When the conserved asparagine is replaced by aspartate, the stability of the mutant proteins decreases by 1.5 and 1.8 kcal/mol for RNases Sa and T1, respectively, but increases by 0.5 kcal/mol for RNase Ba. When the conserved asparagine was replaced by serine, the stability of the mutant proteins was decreased by 2.3 and 1.7 kcal/mol for RNases Sa and T1, respectively. The structure of the Asn 39 --> Ser mutant of RNase Sa was determined at 1.7 A resolution. There is a significant conformational change near the site of the mutation: (1) the side chain of Ser 39 is oriented differently than that of Asn 39 and forms hydrogen bonds with two conserved water molecules; (2) the peptide bond of Ser 42 changes conformation in the mutant so that the side chain forms three new intramolecular hydrogen bonds with the backbone to replace three hydrogen bonds to water molecules present in the wild-type structure; and (3) the loss of the anchoring hydrogen bonds makes the surface loop more flexible in the mutant than it is in wild-type RNase Sa. The results show that burial and hydrogen bonding of the conserved asparagine make a large contribution to microbial RNase stability and emphasize the importance of structural information in interpreting stability studies of mutant proteins.