1ccs
From Proteopedia
(New page: 200px<br /> <applet load="1ccs" size="450" color="white" frame="true" align="right" spinBox="true" caption="1ccs, resolution 2.35Å" /> '''STRUCTURE-ASSISTED ...) |
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| - | [[Image:1ccs.gif|left|200px]]<br /> | + | [[Image:1ccs.gif|left|200px]]<br /><applet load="1ccs" size="350" color="white" frame="true" align="right" spinBox="true" |
| - | <applet load="1ccs" size=" | + | |
caption="1ccs, resolution 2.35Å" /> | caption="1ccs, resolution 2.35Å" /> | ||
'''STRUCTURE-ASSISTED REDESIGN OF A PROTEIN-ZINC BINDING SITE WITH FEMTOMOLAR AFFINITY'''<br /> | '''STRUCTURE-ASSISTED REDESIGN OF A PROTEIN-ZINC BINDING SITE WITH FEMTOMOLAR AFFINITY'''<br /> | ||
==Overview== | ==Overview== | ||
| - | We have inserted a fourth protein ligand into the zinc coordination | + | 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== | ==Disease== | ||
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==About this Structure== | ==About this Structure== | ||
| - | 1CCS is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens] with ZN as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Carbonate_dehydratase Carbonate dehydratase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=4.2.1.1 4.2.1.1] Full crystallographic information is available from [http:// | + | 1CCS is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens] with <scene name='pdbligand=ZN:'>ZN</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Carbonate_dehydratase Carbonate dehydratase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=4.2.1.1 4.2.1.1] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1CCS OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Homo sapiens]] | [[Category: Homo sapiens]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
| - | [[Category: Christianson, D | + | [[Category: Christianson, D W.]] |
| - | [[Category: Ippolito, J | + | [[Category: Ippolito, J A.]] |
[[Category: ZN]] | [[Category: ZN]] | ||
[[Category: lyase (oxo-acid)]] | [[Category: lyase (oxo-acid)]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:04:44 2008'' |
Revision as of 10:04, 21 February 2008
|
STRUCTURE-ASSISTED REDESIGN OF A PROTEIN-ZINC BINDING SITE WITH FEMTOMOLAR AFFINITY
Contents |
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
1CCS is a Single protein structure of sequence from Homo sapiens with as ligand. 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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