1CCU

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

Summary for 1CCU

• Entry DOI: 10.2210/pdb1ccu/pdb
• Descriptor: CARBONIC ANHYDRASE II, ZINC ION, SULFATE ION, ... (4 entities in total)
• Functional Keywords: lyase (oxo-acid)
• Biological source: Homo sapiens (human)
• Cellular location: Cytoplasm : P00918
• Total number of polymer chains: 1
• Total formula weight: 29356.38

Authors

Ippolito, J.A.,Christianson, D.W. (deposition date: 1994-12-09, release date: 1995-02-07, Last modification date: 2024-02-07)

Primary citation

Ippolito, J.A.,Baird Jr., T.T.,McGee, S.A.,Christianson, D.W.,Fierke, C.A.
Structure-assisted redesign of a protein-zinc-binding site with femtomolar affinity.
Proc.Natl.Acad.Sci.USA, 92:5017-5021, 1995

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.

PubMed: 7761440
DOI: 10.1073/pnas.92.11.5017

Experimental method

X-RAY DIFFRACTION (2.25 Å)