PDBsum entry 1kda

Hydrolase (phosphoric diester)

PDB id: 1kda

Contents

Protein chain

137 a.a. *

Waters ×52

* Residue conservation analysis

PDB id:

1kda

Name:

Hydrolase (phosphoric diester)

Title:

Stabilization of a strained protein loop conformation through protein engineering

Structure:

Staphylococcal nuclease. Chain: a. Engineered: yes

Source:

Staphylococcus aureus. Organism_taxid: 1280

Resolution:

1.90Å R-factor: 0.189

Authors:

A.Hodel,R.O.Fox

Key ref:

A.Hodel et al. (1995). Stabilization of a strained protein loop conformation through protein engineering. Protein Sci, 4, 484-495. PubMed id: 7795531 DOI: 10.1002/pro.5560040315

Date:

25-Aug-94 Release date: 27-Feb-95

Protein chain

P00644  (NUC_STAAU) -  Thermonuclease from Staphylococcus aureus

Seq: 231 a.a.

Struc: 137 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.3.1.31.1 - micrococcal nuclease.
• Reaction: Endonucleolytic cleavage to nucleoside 3'-phosphates and 3'-phosphooligonucleotide end-products.

DOI no: 10.1002/pro.5560040315

Protein Sci 4:484-495 (1995)

PubMed id: 7795531

Stabilization of a strained protein loop conformation through protein engineering.

A.Hodel, R.A.Kautz, R.O.Fox.

ABSTRACT

Staphylococcal nuclease is found in two folded conformations that differ in the isomerization of the Lys 116-Pro 117 peptide bond, resulting in two different conformations of the residue 112-117 loop. The cis form is favored over the trans with an occupancy of 90%. Previous mutagenesis studies have shown that when Lys 116 is replaced by glycine, a trans conformation is stabilized relative to the cis conformation by the release of steric strain in the trans form. However, when Lys 116 is replaced with alanine, the resulting variant protein is identical to the wild-type protein in its structure and in the dominance of the cis configuration. The results of these studies suggested that any nuclease variant with a non-glycine residue at position 116 should also favor the cis form because of steric requirements of the beta-carbon at this position. In this report, we present a structural analysis of four nuclease variants with substitutions at position 116. Two variants, K116E and K116M, follow the "beta-carbon" hypothesis by favoring the cis form. Furthermore, the crystal structure of K116E is nearly identical to that of the wild-type protein. Two additional variants, K116D and K116N, provide exceptions to this simple "beta-carbon" rule in that the trans conformation is stabilized relative to the cis configuration by these substitutions. Crystallographic data indicate that this stabilization is effected through the addition of tertiary interactions between the side chain of position 116 with the surrounding protein and water structure. The detailed trans conformation of the K116D variant appears to be similar to the trans conformation observed in the K116G variant, suggesting that these two mutations stabilize the same conformation but through different mechanisms.

Selected figure(s)

Figure 7.
Fig. 7. A: Hydrogen bondingpattern between residues 1 I5 nd 118 of nuclease A (Hynes & Fox, 1991) andthesurrounding ordered water. Residues 115-1 8 adopt a type IVa &turn with a hydrogen bond between the carbonyl oxygen of Tyr 115 and thebackboneamide of Asn 118. B: Hydrogen bonding pattern between residues 115 and 18 of 116D and thesurrounding ordered water. The Asp 116 side chain replaces thebackbone of Tyr 115 n many of the hydrogen bonds observed in the wild- typestructure. C: omparison of the residue 112-118 loopconformationsfromthe K116D variant (white) nd the 116G nuclease variant (Hodel et al., 1992) (green). Oxygen atoms re colored red; nitrogen is colored blue.

Figure 8.
Fig. 8. Electrondensitysurroundingresidues112-1 18 of the K116Nvariant.Shownisthe F, - F SA-omit mapcalculatedusing thenucleaseAstructureexcluding the atoms of residues112-1 18. Mapis contouredat 1.5~. Densityyieldsthe conformation of esidues112-114and116-1 17, ut conformation of residue115isnotclear. Two odels areshownwiththe map. A model similar to hat of K116Dwasbiltandrefined to the data. B Amodelbased on he conformation found in the K116Gvariant is also consistentwiththeelectron density. Although thedensity in the SA-omitmap favors the conformation of 115in the K116G model (B), stericconsiderationssuggestthattheK116D (A) is morelikely to be adopted by K116N.

The above figures are reprinted by permission from the Protein Society: Protein Sci (1995, 4, 484-495) copyright 1995.

Figures were selected by an automated process.