2O36

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


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.95 Å
  • R-Value Free: 0.238 
  • R-Value Work: 0.202 

wwPDB Validation   3D Report Full Report


This is version 1.5 of the entry. See complete history


Literature

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

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

(2007) J Biol Chem 282: 9722-9732

  • DOI: https://doi.org/10.1074/jbc.M609897200
  • Primary Citation of Related Structures:  
    2O36, 2O3E

  • PubMed 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.


  • Organizational Affiliation

    Department of Molecular and Cellular Biochemistry and Center for Structural Biology, University of Kentucky, Lexington, Kentucky 40536.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Thimet oligopeptidase674Homo sapiensMutation(s): 4 
Gene Names: THOP1
EC: 3.4.24.15
UniProt & NIH Common Fund Data Resources
Find proteins for P52888 (Homo sapiens)
Explore P52888 
Go to UniProtKB:  P52888
PHAROS:  P52888
GTEx:  ENSG00000172009 
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP52888
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 1 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
ZN
Query on ZN

Download Ideal Coordinates CCD File 
B [auth A]ZINC ION
Zn
PTFCDOFLOPIGGS-UHFFFAOYSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.95 Å
  • R-Value Free: 0.238 
  • R-Value Work: 0.202 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 77.12α = 90
b = 99.25β = 90
c = 105.71γ = 90
Software Package:
Software NamePurpose
DENZOdata reduction
SCALEPACKdata scaling
CNSrefinement
PDB_EXTRACTdata extraction
HKL-2000data reduction
HKL-2000data scaling
CNSphasing

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2007-01-23
    Type: Initial release
  • Version 1.1: 2008-05-01
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2017-10-18
    Changes: Refinement description
  • Version 1.4: 2021-10-20
    Changes: Database references, Derived calculations
  • Version 1.5: 2023-08-30
    Changes: Data collection, Refinement description