PDBsum entry 1tt2

Hydrolase

PDB id: 1tt2

Contents

Protein chain

130 a.a. *

Ligands

DMS ×2

THP

GOL

Metals

_CA

Waters ×116

* Residue conservation analysis

PDB id:

1tt2

Name:

Hydrolase

Title:

Cryogenic crystal structure of staphylococcal nuclease variant truncated delta+phs i92k

Structure:

Thermonuclease. Chain: a. Synonym: tnase, micrococcal nuclease, staphylococcal nuclease. Engineered: yes. Mutation: yes

Source:

Staphylococcus aureus. Organism_taxid: 1280. Strain: foggi. Gene: nuc. Expressed in: escherichia coli bl21. Expression_system_taxid: 511693.

Resolution:

1.85Å R-factor: 0.189 R-free: 0.211

Authors:

D.M.Nguyen,R.L.Reynald,A.G.Gittis,E.E.Lattman

Key ref:

D.M.Nguyen et al. (2004). X-ray and thermodynamic studies of staphylococcal nuclease variants I92E and I92K: insights into polarity of the protein interior. J Mol Biol, 341, 565-574. PubMed id: 15276844 DOI: 10.1016/j.jmb.2004.05.066

Date:

21-Jun-04 Release date: 06-Jul-04

Protein chain

P00644  (NUC_STAAU) -  Thermonuclease from Staphylococcus aureus

Seq: 231 a.a.

Struc: 130 a.a.*

* PDB and UniProt seqs differ at 6 residue positions (black crosses)

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: 10.1016/j.jmb.2004.05.066

J Mol Biol 341:565-574 (2004)

PubMed id: 15276844

X-ray and thermodynamic studies of staphylococcal nuclease variants I92E and I92K: insights into polarity of the protein interior.

D.M.Nguyen, R.Leila Reynald, A.G.Gittis, E.E.Lattman.

ABSTRACT

We have used crystallography and thermodynamic analysis to study nuclease variants I92E and I92K, in which an ionizable side-chain is placed in the hydrophobic core of nuclease. We find that the energetic cost of burying ionizable groups is rather modest. The X-ray determinations show water molecules solvating the buried glutamic acid under cryo conditions, but not at room temperature. The lysine side-chain does not appear solvated in either case. Guanidine hydrochloride (GnHCl) denaturation of I92E and I92K, done as a function of pH and monitored by tryptophan fluorescence, showed that I92E and I92K are folded in the pH range pH 3.5-9.0 and pH 5.5-9.5, respectively. The stability of the parental protein is independent of pH over a broad range. In contrast, the stabilities of I92E and I92K exhibit a pH dependence, which is quantitatively explained by thermodynamic analysis: the PK(a) value of the buried K92 is 5.6, while that of the buried E92 is 8.65. The free energy difference between burying the uncharged and charged forms of the groups is modest, about 6 kcal/mol. We also found that epsilon(app) for I92K and I92E is in the range approximately 10-12, instead of 2-4 commonly used to represent the protein interior. Side-chains 92E and 92K were uncharged under the conditions of the X-ray experiment. Both are buried completely inside the well-defined hydrophobic core of the variant proteins without forming salt-bridges or hydrogen bonds to other functional groups of the proteins. Under cryo conditions 92E shows a chain of four water molecules, which hydrate one oxygen atom of the carboxyl group of the glutamic acid. Two other water molecules, which are present in the wild-type at all temperatures, are also connected to the water ring observed inside the hydrophobic core. The ready burial of water with an uncharged E92 raises the possibility that solvent excursions into the interior also take place in the wild-type protein, but in a random, dynamic way not detectable by crystallography. Such transient excursions could increase the average polarity, and thus epsilon(app), of the protein interior.

Selected figure(s)

Figure 3.
Figure 3. Ribbon representation of the superposition of the I92E cryo (cyan) and (a) I92E room temperature (red) structures and (b) the I92K cryo structure. (a) The side- chain of 92E in both cryo (blue) and room temperature (orange) adopts an identical conformation. The blue spheres represent the four completely buried and the two bridging water molecules seen in the I92E cryo structure. (b) The two conformations of 92K as observed in the I92K cryo structure with hydrophobic residues that surround conformers 1 (magenta) and conformer 2 (green) within 4 Å .

Figure 5.
Figure 5. The two conformations of 92K built into the 2Fo 2 Fc (blue) and Fo 2 Fc (magenta) electron density maps, contoured at 1s and 3.5s respectively, calculated from a model that had alanine at position 92. The sub- sequent refinement of the model with the alternate 92K conformations showed no electron density for the C d and N z atoms for conformer 1 or for the N z for conformer 2, which is indicative of increased mobility of the 92K side-chain. The electron density maps displayed might suggest the native isoleucine at this position. The lysine mutation was confirmed by DNA sequencing and mass spectrometry (data not shown). Moreover, none of the four most frequent isoleucine conformers could be built into these electron density maps (see Supplementary Material).

The above figures are reprinted by permission from Elsevier: J Mol Biol (2004, 341, 565-574) copyright 2004.

Figures were selected by an automated process.