1CCU

STRUCTURE-ASSISTED REDESIGN OF A PROTEIN-ZINC BINDING SITE WITH FEMTOMOLAR AFFINITY


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.25 Å
  • R-Value Observed: 0.178 

wwPDB Validation   3D Report Full Report


This is version 1.4 of the entry. See complete history


Literature

Structure-assisted redesign of a protein-zinc-binding site with femtomolar affinity.

Ippolito, J.A.Baird Jr., T.T.McGee, S.A.Christianson, D.W.Fierke, C.A.

(1995) Proc Natl Acad Sci U S A 92: 5017-5021

  • DOI: https://doi.org/10.1073/pnas.92.11.5017
  • Primary Citation of Related Structures:  
    1CCS, 1CCT, 1CCU

  • PubMed Abstract: 

    We have inserted a fourth protein ligand into the zinc coordination polyhedron of carbonic anhydrase II (CAII) that increases metal affinity 200-fold (Kd = 20 fM). The three-dimensional structures of threonine-199-->aspartate (T199D) and threonine-199-->glutamate (T199E) CAIIs, determined by x-ray crystallographic methods to resolutions of 2.35 Angstrum and 2.2 Angstrum, respectively, reveal a tetrahedral metal-binding site consisting of H94, H96, H119, and the engineered carboxylate side chain, which displaces zinc-bound hydroxide. Although the stereochemistry of neither engineered carboxylate-zinc interaction is comparable to that found in naturally occurring protein zinc-binding sites, protein-zinc affinity is enhanced in T199E CAII demonstrating that ligand-metal separation is a significant determinant of carboxylate-zinc affinity. In contrast, the three-dimensional structure of threonine-199-->histidine (T199H) CAII, determined to 2.25-Angstrum resolution, indicates that the engineered imidazole side chain rotates away from the metal and does not coordinate to zinc; this results in a weaker zinc-binding site. All three of these substitutions nearly obliterate CO2 hydrase activity, consistent with the role of zinc-bound hydroxide as catalytic nucleophile. The engineering of an additional protein ligand represents a general approach for increasing protein-metal affinity if the side chain can adopt a reasonable conformation and achieve inner-sphere zinc coordination. Moreover, this structure-assisted design approach may be effective in the development of high-sensitivity metal ion biosensors.


  • Organizational Affiliation

    Department of Chemistry, University of Pennsylvania, Philadelphia 19104-6323, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
CARBONIC ANHYDRASE II259Homo sapiensMutation(s): 0 
EC: 4.2.1.1
UniProt & NIH Common Fund Data Resources
Find proteins for P00918 (Homo sapiens)
Explore P00918 
Go to UniProtKB:  P00918
PHAROS:  P00918
GTEx:  ENSG00000104267 
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP00918
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 2 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
SO4
Query on SO4

Download Ideal Coordinates CCD File 
C [auth A]SULFATE ION
O4 S
QAOWNCQODCNURD-UHFFFAOYSA-L
ZN
Query on ZN

Download Ideal Coordinates CCD File 
B [auth A]ZINC ION
Zn
PTFCDOFLOPIGGS-UHFFFAOYSA-N
Binding Affinity Annotations 
IDSourceBinding Affinity
SO4 BindingDB:  1CCU Ki: min: 8.90e+7, max: 3.00e+8 (nM) from 4 assay(s)
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.25 Å
  • R-Value Observed: 0.178 
  • Space Group: P 1 21 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 42.7α = 90
b = 41.7β = 104.6
c = 73γ = 90
Software Package:
Software NamePurpose
PROLSQrefinement

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 1995-02-07
    Type: Initial release
  • Version 1.1: 2008-03-24
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2017-11-29
    Changes: Derived calculations, Other
  • Version 1.4: 2024-02-07
    Changes: Data collection, Database references, Derived calculations