PDBsum entry: 2o3e

Hydrolase

PDB id: 2o3e

Contents

Protein chain

665 a.a. *

Metals

_ZN

Waters ×209

* Residue conservation analysis

PDB id:

2o3e

Name:

Hydrolase

Title:

Crystal structure of engineered neurolysin with thimet oligopeptidase specificity for neurotensin cleavage site.

Structure:

Neurolysin. Chain: a. Synonym: neurotensin endopeptidase, mitochondrial oligopeptidase m, microsomal endopeptidase, mep. Engineered: yes. Mutation: yes

Source:

Rattus norvegicus. Norway rat. Organism_taxid: 10116. Gene: nln. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.20Å R-factor: 0.219 R-free: 0.268

Authors:

D.W.Rodgers,E.J.Lim

Key ref:

E.J.Lim et al. (2007). Swapping the substrate specificities of the neuropeptidases neurolysin and thimet oligopeptidase. J Biol Chem, 282, 9722-9732. PubMed id: 17251185 DOI: 10.1074/jbc.M609897200

Date:

01-Dec-06 Release date: 23-Jan-07

Protein chain

P42676  (NEUL_RAT) -  Neurolysin, mitochondrial

Seq: 704 a.a.

Struc: 665 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.4.24.16 - Neurolysin.
• Reaction: Preferential cleavage in neurotensin: 10-Pro-|-Tyr-11.
• Cofactor: Zinc

 Gene Ontology (GO) functional annotation 

• Cellular component: cytoplasm (3 terms)
• Biological process: proteolysis (1 term)
• Biochemical function: hydrolase activity (5 terms)

DOI no: 10.1074/jbc.M609897200

J Biol Chem 282:9722-9732 (2007)

PubMed id: 17251185

Swapping the substrate specificities of the neuropeptidases neurolysin and thimet oligopeptidase.

E.J.Lim, S.Sampath, J.Coll-Rodriguez, J.Schmidt, K.Ray, D.W.Rodgers.

ABSTRACT

Thimet oligopeptidase (EC 3.4.24.15) and neurolysin (EC 3.4.24.16) are closely related zinc-dependent metallopeptidases that metabolize small bioactive peptides. They cleave many substrates at the same sites, but they recognize different positions on others, including neurotensin, a 13-residue peptide involved in modulation of dopaminergic circuits, pain perception, and thermoregulation. On the basis of crystal structures and previous mapping studies, four sites (Glu-469/Arg-470, Met-490/Arg-491, His-495/Asn-496, and Arg-498/Thr-499; thimet oligopeptidase residues listed first) in their substrate-binding channels appear positioned to account for differences in specificity. Thimet oligopeptidase mutated so that neurolysin residues are at all four positions cleaves neurotensin at the neurolysin site, and the reverse mutations in neurolysin switch hydrolysis to the thimet oligopeptidase site. Using a series of constructs mutated at just three of the sites, it was determined that mutations at only two (Glu-469/Arg-470 and Arg-498/Thr-499) are required to swap specificity, a result that was confirmed by testing the two-mutant constructs. If only either one of the two sites is mutated in thimet oligopeptidase, then the enzyme cleaves almost equally at the two hydrolysis positions. Crystal structures of both two-mutant constructs show that the mutations do not perturb local structure, but side chain conformations at the Arg-498/Thr-499 position differ from those of the mimicked enzyme. A model for differential recognition of neurotensin based on differences in surface charge distribution in the substrate binding sites is proposed. The model is supported by the finding that reducing the positive charge on the peptide results in cleavage at both hydrolysis sites.

Selected figure(s)

Figure 5.
FIGURE 5. Comparison of the substituted residues in TOP2 and neurolysin 2 with the corresponding residues in wild type neurolysin and TOP. A, the side chain of Arg-469 from TOP2 (cyan) with Arg-470 from wild type neurolysin (green). Backbone atoms are shown in a worm representation. B, Thr-498 from TOP2 (cyan) and Thr-499 from neurolysin (green). C, conformational differences between TOP2 (cyan) and neurolysin (green) in a loop near the Thr-498/499 position. In addition to Thr-498/499, residues 599–611 of TOP2 and residues 600–612 of neurolysin are shown in a backbone worm representation. The side chains of Tyr-605/606 in the two structures are also shown. D, Glu-470 from neurolysin 2 (green) and Glu-469 from wild type neurolysin (cyan). E, Arg-499 from neurolysin 2 and Arg-498 from wild type TOP. F, loop region in neurolysin 2 (green) and wild type TOP (cyan) near Arg-499/498. Residue ranges are the same as in C.

Figure 7.
FIGURE 7. Model for differential specificity of TOP and neurolysin with respect to primary NT hydrolysis sites. A, cut away molecular surface views of the TOP (left) and neurolysin (right) binding sites colored according to surface electrostatic potential (red, negative; blue, positive). The active site zinc ion is shown as a blue sphere. Schematic representations of the NT peptide in two binding registrations emphasizing the positively charged region in the center of the peptide are also shown. B, schematic NT representations with the key residues mediating differential specificity shown along with their contribution to surface electrostatic potential in the substrate binding site. C, similar representation of the NT(R9E) peptide with the key residues in wild type TOP and the TOP(E469R) mutant.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2007, 282, 9722-9732) copyright 2007.

Figures were selected by an automated process.