PDBsum entry 1ccs

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protein metals links
Lyase (oxo-acid) PDB id
Protein chain
255 a.a. *
Waters ×122
* Residue conservation analysis
PDB id:
Name: Lyase (oxo-acid)
Title: Structure-assisted redesign of a protein-zinc binding site with femtomolar affinity
Structure: Carbonic anhydrase ii. Chain: a. Engineered: yes
Source: Homo sapiens. Human. Organism_taxid: 9606
2.35Å     R-factor:   0.178    
Authors: J.A.Ippolito,D.W.Christianson
Key ref: J.A.Ippolito et al. (1995). Structure-assisted redesign of a protein-zinc-binding site with femtomolar affinity. Proc Natl Acad Sci U S A, 92, 5017-5021. PubMed id: 7761440 DOI: 10.1073/pnas.92.11.5017
09-Dec-94     Release date:   07-Feb-95    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
P00918  (CAH2_HUMAN) -  Carbonic anhydrase 2
260 a.a.
255 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 1 residue position (black cross)

 Enzyme reactions 
   Enzyme class: E.C.  - Carbonate dehydratase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: H2CO3 = CO2 + H2O
= CO(2)
+ H(2)O
      Cofactor: Zn(2+)
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     extracellular space   11 terms 
  Biological process     angiotensin-mediated signaling pathway   22 terms 
  Biochemical function     protein binding     5 terms  


    Added reference    
DOI no: 10.1073/pnas.92.11.5017 Proc Natl Acad Sci U S A 92:5017-5021 (1995)
PubMed id: 7761440  
Structure-assisted redesign of a protein-zinc-binding site with femtomolar affinity.
J.A.Ippolito, T.T.Baird, S.A.McGee, D.W.Christianson, C.A.Fierke.
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.

Literature references that cite this PDB file's key reference

  PubMed id Reference
19818877 T.K.Hurst, D.Wang, R.B.Thompson, and C.A.Fierke (2010).
Carbonic anhydrase II-based metal ion sensing: Advances and new perspectives.
  Biochim Biophys Acta, 1804, 393-403.  
18027003 L.A.Yatsunyk, J.A.Easton, L.R.Kim, S.A.Sugarbaker, B.Bennett, R.M.Breece, I.I.Vorontsov, D.L.Tierney, M.W.Crowder, and A.C.Rosenzweig (2008).
Structure and metal binding properties of ZnuA, a periplasmic zinc transporter from Escherichia coli.
  J Biol Inorg Chem, 13, 271-288.
PDB codes: 2prs 2ps0 2ps3 2ps9
18335973 V.M.Krishnamurthy, G.K.Kaufman, A.R.Urbach, I.Gitlin, K.L.Gudiksen, D.B.Weibel, and G.M.Whitesides (2008).
Carbonic anhydrase as a model for biophysical and physical-organic studies of proteins and protein-ligand binding.
  Chem Rev, 108, 946.  
18020665 M.V.Inamdar, C.M.Lastoskie, C.A.Fierke, and A.M.Sastry (2007).
Mobile trap algorithm for zinc detection using protein sensors.
  J Chem Phys, 127, 185102.  
16688707 A.Fernández-Gacio, A.Codina, J.Fastrez, O.Riant, and P.Soumillion (2006).
Transforming carbonic anhydrase into epoxide synthase by metal exchange.
  Chembiochem, 7, 1013-1016.  
12056894 S.Huang, B.Sjöblom, A.E.Sauer-Eriksson, and B.H.Jonsson (2002).
Organization of an efficient carbonic anhydrase: implications for the mechanism based on structure-function studies of a T199P/C206S mutant.
  Biochemistry, 41, 7628-7635.
PDB codes: 1lg5 1lg6 1lgd
11316870 J.D.Cronk, J.A.Endrizzi, M.R.Cronk, J.W.O'neill, and K.Y.Zhang (2001).
Crystal structure of E. coli beta-carbonic anhydrase, an enzyme with an unusual pH-dependent activity.
  Protein Sci, 10, 911-922.
PDB codes: 1i6o 1i6p
9751639 D.E.Benson, M.S.Wisz, and H.W.Hellinga (1998).
The development of new biotechnologies using metalloprotein design.
  Curr Opin Biotechnol, 9, 370-376.  
9538005 D.W.Pettigrew, N.D.Meadow, S.Roseman, and S.J.Remington (1998).
Cation-promoted association of Escherichia coli phosphocarrier protein IIAGlc with regulatory target protein glycerol kinase: substitutions of a Zinc(II) ligand and implications for inducer exclusion.
  Biochemistry, 37, 4875-4883.  
9586241 H.W.Hellinga, and J.S.Marvin (1998).
Protein engineering and the development of generic biosensors.
  Trends Biotechnol, 16, 183-189.  
9020770 J.Guo, and D.P.Giedroc (1997).
Zinc site redesign in T4 gene 32 protein: structure and stability of cobalt(II) complexes formed by wild-type and metal ligand substitution mutants.
  Biochemistry, 36, 730-742.  
9241423 J.M.Berg, and H.A.Godwin (1997).
Lessons from zinc-binding peptides.
  Annu Rev Biophys Biomol Struct, 26, 357-371.  
8639494 C.C.Huang,, C.A.Lesburg, L.L.Kiefer, C.A.Fierke, and D.W.Christianson (1996).
Reversal of the hydrogen bond to zinc ligand histidine-119 dramatically diminishes catalysis and enhances metal equilibration kinetics in carbonic anhydrase II.
  Biochemistry, 35, 3439-3446.
PDB codes: 1zsa 1zsb 1zsc
8768904 H.W.Hellinga (1996).
Metalloprotein design.
  Curr Opin Biotechnol, 7, 437-441.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.