PDBsum entry 1ccs

Lyase (oxo-acid)

PDB id: 1ccs

Contents

Protein chain

255 a.a. *

Metals

_ZN

Waters ×122

* Residue conservation analysis

PDB id:

1ccs

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

Resolution:

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

Date:

09-Dec-94 Release date: 07-Feb-95

Protein chain

P00918  (CAH2_HUMAN) -  Carbonic anhydrase 2 from Homo sapiens

Seq: 260 a.a.

Struc: 255 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class 2: E.C.4.2.1.1 - carbonic anhydrase.
• Reaction: hydrogencarbonate + H⁺ = CO2 + H2O
• Cofactor: Zn(2+)
• Enzyme class 3: E.C.4.2.1.69 - cyanamide hydratase.
• Reaction: urea = cyanamide + H2O

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

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.

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.