PDBsum entry 2rdf

Hydrolase

PDB id: 2rdf

Contents

Protein chain

125 a.a. *

Ligands

PO4

Waters ×72

* Residue conservation analysis

PDB id:

2rdf

Name:

Hydrolase

Title:

Crystal structure of staphyloccocal nuclease viagan/e75a variant at cryogenic temperature

Structure:

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

Source:

Staphylococcus aureus. Gene: nuc. Expressed in: escherichia coli.

Resolution:

2.01Å R-factor: 0.218 R-free: 0.244

Authors:

K.Baran,J.L.Schlessman,B.E.Garcia-Moreno

Key ref:

K.L.Baran et al. (2008). Electrostatic effects in a network of polar and ionizable groups in staphylococcal nuclease. J Mol Biol, 379, 1045-1062. PubMed id: 18499123 DOI: 10.1016/j.jmb.2008.04.021

Date:

22-Sep-07 Release date: 10-Jun-08

Protein chain

P00644  (NUC_STAAU) -  Thermonuclease from Staphylococcus aureus

Seq: 231 a.a.

Struc: 125 a.a.*

* PDB and UniProt seqs differ at 7 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.2008.04.021

J Mol Biol 379:1045-1062 (2008)

PubMed id: 18499123

Electrostatic effects in a network of polar and ionizable groups in staphylococcal nuclease.

K.L.Baran, M.S.Chimenti, J.L.Schlessman, C.A.Fitch, K.J.Herbst, B.E.Garcia-Moreno.

ABSTRACT

His121 and His124 are embedded in a network of polar and ionizable groups on the surface of staphylococcal nuclease. To examine how membership in a network affects the electrostatic properties of ionizable groups, the tautomeric state and the pK(a) values of these histidines were measured with NMR spectroscopy in the wild-type nuclease and in 13 variants designed to disrupt the network. In the background protein, His121 and His124 titrate with pK(a) values of 5.2 and 5.6, respectively. In the variants, where the network was disrupted, the pK(a) values range from 4.03 to 6.46 for His121, and 5.04 to 5.99 for His124. The largest decrease in a pK(a) was observed when the favorable Coulomb interaction between His121 and Glu75 was eliminated; the largest increase was observed when Tyr91 or Tyr93 was substituted with Ala or Phe. In all variants, the dominant tautomeric state at neutral pH was the N(epsilon2) state. At one level the network behaves as a rigid unit that does not readily reorganize when disrupted: crystal structures of the E75A or E75Q variants show that even when the pivotal Glu75 is removed, the overall configuration of the network was unaffected. On the other hand, a few key hydrogen bonds appear to govern the conformation of the network, and when these bonds are disrupted the network reorganizes. Coulomb interactions within the network report an effective dielectric constant of 20, whereas a dielectric constant of 80 is more consistent with the magnitude of medium to long-range Coulomb interactions in this protein. The data demonstrate that when structures are treated as static, rigid bodies, structure-based pK(a) calculations with continuum electrostatics method are not useful to treat ionizable groups in cases where pK(a) values are governed by short-range polar and Coulomb interactions.

Selected figure(s)

Figure 1.
Fig. 1. (a) Location of the network surrounding His121 and His124 in the structure of wild-type SNase. (b) A diagram of the network of interactions around His121 and His124. The distances that are shown identify the noncovalent interactions that are most consistent with the experimental data.

Figure 7.
Fig. 7. (a) 2F[o]–F[c] electron density maps contoured around significant amino acid side chains in the E75A variant at 1.25 down triangle, open (light blue). The side chains of Ala75, His121 and His124 are shown in blue and labeled. The neighboring Lys9, Glu73, Asp77, Tyr91, Tyr93, Glu101, Lys127 and Ser128 are shown in yellow. (b) The same as a but with the structure of the E75Q variant. (c) Backbone superposition of wild-type SNase (gray) (PDB accession code 1stn.pbd), E75A (blue) and E75Q (red). (d) Stereoview of the superposition of wild-type SNase (gray), E75A (blue) and E75Q (red). Side chains of Lys7, Glu73, Asp77, Tyr91, and Tyr93 are shown for comparison. The C^α RMSD of the structure of E75A or E75Q against the wild type was 0.60 Å.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2008, 379, 1045-1062) copyright 2008.

Figures were selected by an automated process.