PDBsum entry 1ccs

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Lyase (oxo-acid) PDB id
Protein chain
255 a.a.
Waters ×122

References listed in PDB file
Key reference
Title Structure-Assisted redesign of a protein-Zinc-Binding site with femtomolar affinity.
Authors J.A.Ippolito, T.T.Baird, S.A.Mcgee, D.W.Christianson, C.A.Fierke.
Ref. Proc Natl Acad Sci U S A, 1995, 92, 5017-5021. [DOI no: 10.1073/pnas.92.11.5017]
PubMed id 7761440
Note In the PDB file this reference is annotated as "TO BE PUBLISHED". The citation details given above were identified by an automated search of PubMed on title and author names, giving a percentage match of 88%.
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.
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