1ccu

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spinqualitylabelsall models 1ccu, resolution 2.25Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

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

Contents 1 Overview 2 Disease 3 About this Structure 4 Reference

Overview

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.

Disease

Known disease associated with this structure: Osteopetrosis, autosomal recessive 3, with renal tubular acidosis OMIM:[611492]

About this Structure

1CCU is a Single protein structure of sequence from Homo sapiens with ZN and SO4 as ligands. Active as Carbonate dehydratase, with EC number 4.2.1.1 Full crystallographic information is available from OCA.

Reference

Structure-assisted redesign of a protein-zinc-binding site with femtomolar affinity., Ippolito JA, Baird TT Jr, McGee SA, Christianson DW, Fierke CA, Proc Natl Acad Sci U S A. 1995 May 23;92(11):5017-21. PMID:7761440

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