ArticleProceedings of the National Academy of Sciences of the United States of America1995
Structure-assisted redesign of a protein-zinc-binding site with femtomolar affinity.
Article in Proceedings of the National Academy of Sciences of the United States of America, 1995. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 15 papers.
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Who cites it
15 citing papers in PubMed, 65 citations in OpenAlex.
- Conformational flexibility of His200 enables catalytic activity in the T200H mutant of carbonic anhydrase II.Molecules and cells · 2025Article
- Metal3D: a general deep learning framework for accurate metal ion location prediction in proteins.Nature communications · 2023Article
- Metallo-β-lactamases in the Age of Multidrug Resistance: From Structure and Mechanism to Evolution, Dissemination, and Inhibitor Design.Chemical reviews · 2021Review
- Principles and practice of determining metal-protein affinities.The Biochemical journal · 2021Review
- Article
- Carbonic anhydrase modification for carbon management.Environmental science and pollution research international · 2020Article
- Kinetic Study of COBiomimetics (Basel, Switzerland) · 2019Article
- The Loss and Gain of Functional Amino Acid Residues Is a Common Mechanism Causing Human Inherited Disease.PLoS computational biology · 2016Article
- Protein design: toward functional metalloenzymes.Chemical reviews · 2014Review
- Designing hydrolytic zinc metalloenzymes.Biochemistry · 2014Review
- X-ray structure and mutational analysis of the atrazine Chlorohydrolase TrzN.The Journal of biological chemistry · 2010Article
- Carbonic anhydrase II-based metal ion sensing: Advances and new perspectives.Biochimica et biophysica acta · 2010Review
- Carbonic anhydrase as a model for biophysical and physical-organic studies of proteins and protein-ligand binding.Chemical reviews · 2008Review
- Structure and metal binding properties of ZnuA, a periplasmic zinc transporter from Escherichia coli.Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry · 2008Article
- Crystal structure of E. coli beta-carbonic anhydrase, an enzyme with an unusual pH-dependent activity.Protein science : a publication of the Protein Society · 2001Article
Corrections and comments
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Authors and funding
5 authors at 2 institutions in 1 country.
Funding
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.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.